Pharmaceutical composition comprising plasminogen and uses thereof

ABSTRACT

Pharmaceutical compositions comprising plasminogen or a biologically active variant thereof are disclosed. In an embodiment, the composition comprises a tonicity modifier, a bulking agent, and a stabilising agent and has a pH of about 3.0 to about 10.0. In another embodiment, the composition contains an amount of particles in suspension equal to or greater than 10 μm which is lower than 6000 particles per 100 ml, and preferably lower than 2000 particles per 100 ml. Uses of these compositions as a medicament is contemplated. Various therapeutic uses of these pharmaceutical compositions is also contemplated.

CROSS REFERENCE TO A RELATED APPLICATION

This application is a Continuation Application of co-pending applicationSer. No. 15/536,877, filed Jun. 16, 2017 (now U.S. Pat. No. 10,441,639);which is a National Stage Application of International ApplicationNumber PCT/CA2015/000606, filed Dec. 18, 2015; which claims priority toU.S. Provisional Application No. 62/094,556, filed Dec. 19, 2014; all ofwhich are incorporated herein by reference in their entirety.

The Sequence Listing for this application is labeled“SeqList-30Sep19-ST25.txt”, which was created on Sep. 30, 2019, and is 8KB. The entire content is incorporated herein by reference in itsentirety.

FIELD OF INVENTION

The present invention relates to the field of medicine. Moreparticularly, the invention relates to a pharmaceutical compositioncomprising plasminogen, and therapeutic uses thereof.

BACKGROUND OF INVENTION

Plasminogen is a zymogen of plasmin as shown in FIG. 1A. The amino acidsequence of the human plasminogen precursor is depicted in FIG. 1B.Human plasminogen contains 791 amino acids (precursor=810 amino acids)with a molecular weight of about 90 kDa and a pI of approximately 7.0,although differential glycosylation and/or removal of the N-terminalactivation peptide can result in a pI range of 6.2 to 8.0. It is asingle-chain protein with 24 intra-chain disulfide bridges, 5 kringledomains (involved in the binding to fibrin and to the inhibitorα2-antiplasmin), a serine protease domain (P), and an activation peptide(AP) consisting of the first 77 amino acids. There is one N-linkedglycosylation site and one O-linked site, although a second O-linkedsite has been identified (Goldberg, 2006). Approximately 70% of thePlasminogen in circulation contains only O-linked glycosylation whilethe rest contains both N- and O-linked sugars.

Native Plasminogen is produced in two main forms, Glu-Plasminogen(Glu-Pg) and Lys-Plasminogen (Lys-Pg), named for the N-terminal aminoacid of either glutamic acid or lysine. Glu-Pg is composed of the entireamino acid sequence designated by the gene sequence (excluding theactivation peptide), while Lys-Pg is the result of a cleavage of theGlu-Pg between Lys-77 and Lys-78 (underlined in FIG. 1B). Thecirculating half-life of Lys-Pg is considerably shorter than Glu-Pg(2-2.5 days for Glu-Pg, 0.8 days for Lys-Pg). Glu-Pg is the dominantform of Pg present in plasma with very little Lys-Pg detected in thecirculation (Violand, B. N., Byrne, R., Castellino F. J. (1978) Theeffect of α-,ω-Amino Acids on Human Plasminogen Structure andActivation. J Biol Chem. 253 (15): 5395-5401; Collen D, Ong E B, JohnsonA J. (1975) Human Plasminogen: In Vitro and In Vivo Evidence for theBiological Integrity of NH₂—Terminal Glutamic Acid Plasminogen.Thrombosis Research. 7 (4):515-529).

Plasminogen is synthesized in the liver and secreted into plasma.Plasminogen is distributed throughout the body and when conditions arepresent for activation, the plasminogen pro-enzyme is converted to theactive enzyme, plasmin, by tissue-type plasminogen activator (t-PA) orby urokinase plasminogen activator (u-PA). Plasmin then degrades fibrinand converts latent matrix metalloproteinases (pro-MMPs) into activeMMPs, which in turn further degrade extracellular matrix (ECM) as partof the tissue healing/remodeling process. Plasminogen activationmediated by t-PA is primarily involved in fibrin homeostasis, whileplasmin generation via u-PA, forming a complex with its receptor u-PAR,plays a role in tissue remodeling.

Plasmin is or was investigated for its potential use for the clearanceof thrombotic occlusions in artificial devices and hemodialysis grafts,and for the treatment of posterior vitreous detachment (PVD) (U.S. Pat.No. 6,969,515; US 2010/0104551).

Plasminogen is investigated for its use in therapeutic indications suchas wound healing, healing of a tympanic membrane perforation, healing ofperiodontal wound, infectious disease, oral health, diabetic ulcer,thrombolysis indications, such as coronary thrombosis, reperfusioninjury to tissue, ischemia, infarction, brain edema, improvement ofmicrocirculation, and modulation of complement pathway (U.S. Pat. Nos.8,637,010; 8,679,482; 8,318,661; WO 95/12407; EP 0,631,786). As yet, noplasminogen is currently on the pharmaceutical market as a drug.

Historically, Lys-Pg was pharmaceutically commercialized for a period oftime for hematologic purposes and has not been scientifically ormedically used since 2000. A formulation of Lys-Pg is described inSchott et al., 1998, The New England Journal of Medicine, Vol. 339, No.23, pp. 1679-1686). While available Lys-Pg was clinically used andinvestigated for the treatment of ligneous conjunctivitis, a clinicalmanifestation of the underlying condition hypoplasminogenimia(plasminogen deficiency type I). Hence systemic administration of Lys-Pgconcentrates has been tested. Kraft et al. (Kraft J, Lieb W, Zeitler P,Schuster V. (2000) Ligneous conjunctivitis in a girl with severe type Iplasminogen deficiency. Graefes Arch Clin Exp Ophthalmol.238(9):797-800) reported that daily infusion of Lys-Pg in a child withsevere hypoplasminogenemia resulted in partial resolution of theconjunctival pseudo membranes. Schott et al. (1998) reported that, in a6-month-old child, treatment with Lys-Pg preparation as a continuousinfusion and later as daily bolus injections led to complete regressionof the ligneous conjunctivitis within 4 weeks and normalizedhyperviscous secretions in the respiratory tract as well as skin woundhealing. Schuster et al (Schuster V, Hugle B, Tefs K (2007) Plasminogendeficiency. J Thromb Haemost 5(12):2315-2322) reported that plasminogenlevels in patients with homozygous or compound heterozygous (thepresence of two different mutant alleles at a particular gene locus, oneon each chromosome of a pair) hypoplasminogenemia ranged from <1 to 9mg/dL for plasminogen antigen plasma levels and <1% to 51% forfunctional plasminogen activity. It is important to note that themajority of these patients have some residual plasminogen activitylevels. Thus, plasminogen replacement is expected to be effective, as itis an endogenous protein and is not expected to have immunogenicity orfibrinolytic activity concerns. Although systemic or topical plasminogenconcentrates have been clearly documented as effective therapy leadingto resolution and halts re-formation of the lesions (Watts P, Suresh P,Mezer E, Ells A, Albisetti M, Bajzar L, Marzinotto V, Andrew M,Massicotle P, Rootman D (2002) Effective treatment of ligneousconjunctivitis with topical plasminogen. Am J Ophthalmol 133(4):451-455,Heidemann D G, Williams G A, Hartzer M, Ohanian A, Citron M E (2003)Treatment of ligneous conjunctivitis with topical plasmin and topicalplasminogen. Cornea 22(8):760-762; and Schott, 1998), no purifiedplasminogen product for topical or for systemic therapy is commerciallyavailable.

Only very recently, clinical trials were undertaken to utilize Glu-Pgfor the treatment of the type I plasminogen deficiency (ClinicalTrials.gov Identifier: NCT02312180) and one of its clinicalmanifestation ligneous conjunctivitis utilizing a localized eye drop ofGlu-Pg (ClinicalTrials.gov Identifier: NCT01554956).

Proteins may be stabilized either by changing their structuralcharacteristics (internally) and/or by controlling the components incontact with them (externally). Some proteins have raised particularchallenges with respect to handling and behaviour in pharmaceuticalformulations due to their physico-chemical characteristics which oftenunfortunately leads to structural instability. They can actually undergovarious types of degradations as exemplified by the following: 1)chemical processes leading to the formation of related impurities, whichmay involve hydrolysis, oxidation reactions, deamidation or structuralrearrangements such as iso-asp or intramolecular truncations or 2) aphysical process giving rise to aggregation/polymerization thusgenerating structural alterations which may impact on the biologicalactivity and potentially enhance immunogenicity.

Formulation development generally refers to a process in which an activepharmaceutical ingredient (API) is characterized to a sufficient extentthat it can be converted to a pharmaceutically acceptable drugsubstance. Biophysical characterization of drug substances must beperformed to confirm that the correctly folded and biologically activestructure is present. Several spectroscopic techniques (Fluorescence,CD, DSC, DLS, etc.) can be used to examine the tertiary structure ofproteins in solution and to assess the stability and effect of differentformulations conditions on protein structure. (Volkin, D. B. et al.“Preformulation studies as an essential guide to formulation developmentand manufacture of protein pharmaceutical.” Development and manufactureof protein pharmaceuticals. Edited by Steve L. Nail and Michael J.Akers, Kluwer Academic 2002 Chapter 1, page 1-39; Cheng, W. et al.“Comparison of High-Throughput Biophysical Methods to IdentifyStabilizing Excipients for a Model IgG2 Monoclonal Antibody:Conformational Stability and Kinetic Aggregation Measurements” Journalof Pharmaceutical Sciences, Vol. 101, No 5, page 1701-1720, 2012). Inaddition, often the pharmaceutical industry confirms the structuralintegrity of the API by performing a biologically assay for the releaseof the drug substance. Taken together they confirm that the preferredformulation has not inadvertently been modified by the formulation.Plasminogen is a protein that is mainly used in the preparation ofplasmin for thrombolysis indications, such as coronary thrombosis,clearance of thrombotic occlusions in artificial devices andhemodialysis grafts, and for reperfusion injury to tissue, treatingischemia, infraction, brain edema, or for improving the microcirculation(WO 95/12407; U.S. Pat. No. 6,969,515; EP 0,631,786).

The administration of plasminogen has also been found useful in manytherapeutic indications such as thrombolysis indications, such ascoronary thrombosis, treating reperfusion injury to tissue, treatingischemia, infarction, brain edema, or for improving themicrocirculation, wound healing, healing of a tympanic membraneperforation, healing of periodontal wound, infectious disease, oralhealth, diabetic ulcer, plasminogen-deficient subjects, and modulationof complement pathway (WO 95/12407; EP 0,631,786 U.S. Pat. Nos.8,637,010; 8,679,482; 8,318,661).

There are several challenges encountered when formulating plasminogen.Some problems result from the plasmin contamination of the plasminogenformulation, which degrades plasminogen. Various approaches have beenused for avoiding degradation of plasminogen including the addition ofaprotinin, lysine, phenylmethanesulphonyl fluoride, soybean trypsininhibitor or serine protease inhibitor (U.S. Pat. Nos. 4,177,262;4,361,653, 5,304,383).

Other difficulties are represented by the turbidity or the presence offilamentous substances when plasminogen is solubilised in an aqueoussolvent. To overcome this drawback, it has been proposed to combineplasminogen for example with a nonionic surfactant or with a mixture ofsucrose, amino acid and albumin (WO 94/15631).

There is no known commercial formulation of Glu-Pg for human therapeuticuse. The only formulations of Glu-Pg that are known are for researchonly.

There is a need for the development of a pharmaceutical composition ofplasminogen.

The present description refers to a number of documents, the content ofwhich is herein incorporated by reference in their entirety.

BRIEF SUMMARY OF THE INVENTION

The present invention relates to a pharmaceutical composition comprisingplasminogen, and uses thereof.

The present invention relates to the following items 1 to 61:

1. A pharmaceutical composition comprising:

-   -   plasminogen, or a biologically active variant thereof;    -   a tonicity modifier; and    -   a stabilising agent;    -   wherein the composition has a pH of about 3.0 to about 10.0.

2. The pharmaceutical composition of item 1, wherein the pH is from 5.0to 8.0; or from 6.0 to 8.0; or from 6.5 to 7.5.

3. The pharmaceutical composition of item 1, wherein the concentrationof the plasminogen or biologically active variant thereof is about 0.01mg/ml to about 80 mg/ml, or from about 5 mg/ml to about 60 mg/ml.

4. The pharmaceutical composition of item 3, wherein the concentrationof the plasminogen or biologically active variant thereof is about 40,30, 20, 10 or 5 mg/ml.

5. The pharmaceutical composition of any one of items 1 to 4, whereinthe plasminogen or biologically active variant thereof represents atleast 80% of the total protein content of the composition or more than90% of the total protein content of the composition.

6. The pharmaceutical composition of item 5, wherein the plasminogen orbiologically active variant thereof represents more than 95 or 98% ofthe total protein content of the composition.

7. The pharmaceutical composition of any one of items 1 to 6, whereinthe stabilising agent comprises (i) an amino acid, (ii) an amino acidsalt, (ii) an amino acid analog, or (iv) any mixture of (i), (ii) and/or(iii).

8. The pharmaceutical composition of item 7, wherein the amino acid,amino acid salt or amino acid analog is arginine, proline, glutamicacid, aspartic acid, glycine, alanine, cysteine, lysine, a lysine analogor epsilon-amino caproic acid.

9. The pharmaceutical composition of item 8, wherein the stabilisingagent is arginine.

10. The pharmaceutical composition of any one of items 1 to 9, whereinthe stabilising agent is in a concentration of about 20 mM to about 200mM.

11. The pharmaceutical composition of item 10, wherein the stabilisingagent is in a concentration of about 25 mM to about 75 mM.

12. The pharmaceutical composition of any one of items 1 to 11, whereinthe tonicity modifier is sodium chloride, calcium chloride, magnesiumchloride, sucrose, trehalose, sorbitol, mannitol, glycerol, lactose,sorbitol, dextrose, cyclodextrin, raffinose, polyethylene glycol,hydroxyethyl starch, glycine glutamic acid, alanine, dextran, PVP (polyvinyl pyrrolidone), or any combination thereof.

13. The pharmaceutical composition of item 12, wherein the tonicitymodifier is sodium chloride.

14. The pharmaceutical composition of any one of items 1 to 13, whereinthe tonicity modifier is present in a concentration of about 30 mM toabout 250 mM.

15. The pharmaceutical composition of item 14, wherein the tonicitymodifier is present in a concentration of between about 25 mM to about50 mM, or of about 35 mM.

16. The pharmaceutical composition of any one of items 1 to 15, furthercomprising a bulking agent.

17. The pharmaceutical composition of item 16, wherein the bulking agentis glycine, glutamic acid, alanine, mannitol, sorbitol, hydroxyethylstarch, dextran, PVP (poly vinyl pyrrolidone), mannitol, polyethyleneglycol, sorbitol, maltitol, lactitol, maltotriitol, xylitol or anycombination thereof.

18. The pharmaceutical composition of item 17, wherein the bulking agentis mannitol or sorbitol.

19. The pharmaceutical composition of any one of items 16 to 18, whereinthe bulking agent is in a concentration of about 50 mM to about 300 mM.

20. The pharmaceutical composition of item 19, wherein the bulking agentis in a concentration of about 50 to about 125 mM.

21. The pharmaceutical composition of any one of items 1 to 20, furthercomprising a reducing sugar.

22. The pharmaceutical composition of item 21, wherein the reducingsugar is fructose, mannose, maltose, lactose, arabinose, xylose, ribose,rhamnose, galactose, glucose, sucrose or any combination thereof.

23. The pharmaceutical composition of any one of items 1 to 22, furthercomprising a non-reducing sugar.

24. The pharmaceutical composition of item 23, wherein the non-reducingsugar is sucrose, trehalose, sorbose, melezitose, raffinose, or anycombination thereof.

25. The pharmaceutical composition of any one of items 1 to 24, whereinthe pharmaceutical composition has an osmolality between about 180 mOsmand about 350 mOsm.

26. The pharmaceutical composition of any one of items 1 to 25, furthercomprising a preservative.

27. The pharmaceutical composition of item 26, wherein the preservativeis m-cresol, benzyl alcohol, methanol, ethanol, iso-propanol, butylparaben, ethyl paraben, methyl paraben, phenol, glycerol, xylitol,resorcinol, catechol, 2,6-dimethylcyclohexanol, 2-methyl-2,4-pentadiol,dextran, polyvinylpyrrolidone, 2-chlorophenol, benzethonium chloride,merthiolate (thimersosal), benzoic acid (propyl paraben) MW 180.2,benzoic acid MW 122.12, benzalkonium chloride, chlorobutanol, sodiumbenzoate, sodium propionate, cetylpyridinium chloride, or anycombination thereof.

28. The pharmaceutical composition of item 26 or 27, wherein theconcentration of the preservative is from about 0.005 to about 10%(w/v).

29. The pharmaceutical composition of any one of items 1 to 28, whereinsaid plasminogen or a biologically active variant thereof is humanplasminogen.

30. The pharmaceutical composition of any one of items 1 to 29, whereinsaid plasminogen or a biologically active variant thereof is constitutedof more than about 80% of Glu-plasminogen.

31. The pharmaceutical composition of any one of items 1 to 29, whereinsaid plasminogen or a biologically active variant thereof isGlu-plasminogen.

32. The pharmaceutical composition of any one of items 1 to 31, whereinthe composition contains an amount of particles equal to or greater than10 μm which is lower than 6000 particles per 100 ml.

33. The pharmaceutical composition of item 32, wherein the amount ofparticles is lower than 2000 or 1000 particles per 100 ml.

34. The pharmaceutical composition of any one of items 1 to 33, which issuitable for intravenous, subcutaneous, topical, intradermal, ophthalmicand/or intramuscular administration.

35. The pharmaceutical composition of any one of items 1 to 34, which isa liquid composition, a liquid composition suitable for lyophilisation,a liquid composition suitable for freezing, a lyophilised composition, afrozen composition or a reconstituted composition.

36. The pharmaceutical composition of any one of items 1 to 34, which isa liquid, a gel, a cream, or an ointment.

37. The pharmaceutical composition of any one of items 1 to 36, for useas a medicament.

38. A pharmaceutical composition comprising plasminogen or abiologically active variant thereof, which contains an amount ofparticles equal to or greater than 10 μm which is lower than 6000particles per 100 ml.

39. The pharmaceutical composition of item 38, wherein the amount ofparticles is lower than 2000 or 1000 particles per 100 ml.

40. The pharmaceutical composition of item 38 or 39, which furthercomprises a stabilising agent.

41. The pharmaceutical composition of item 40, wherein the stabilisingagent comprises (i) an amino acid, (ii) an amino acid salt, (ii) anamino acid analog, or (iv) any mixture of (i), (ii) and/or (iii).

42. The pharmaceutical composition of item 41, wherein the amino acid,amino acid salt or amino acid analog is arginine, proline, glutamicacid, aspartic acid, glycine, alanine, cysteine, lysine, a lysine analogor epsilon-amino caproic acid.

43. The pharmaceutical composition of item 42, wherein the stabilisingagent is arginine.

44. The pharmaceutical composition of any one of items 40 to 43, whereinthe stabilising agent is in a concentration of about 25 mM to about 75mM.

45. The pharmaceutical composition of any one of items 38 to 44, whereinthe concentration of the plasminogen or biologically active variantthereof is from about 0.01 mg/ml to about 80 mg/ml.

46. The pharmaceutical composition of item 45, wherein the concentrationof the plasminogen or biologically active variant thereof is from about5 mg/ml to about 60 mg/ml.

47. The pharmaceutical composition of any one of items 38 to 46, whereinthe plasminogen or biologically active variant thereof represents morethan about 95% of the total protein content of the composition, or morethan about 98% of the total protein content of the composition

48. The pharmaceutical composition of any one of items 38 to 47, whichfurther comprises a tonicity modifier.

49. The pharmaceutical composition of item 48, wherein the tonicitymodifier is sodium chloride, calcium chloride, magnesium chloride,sucrose, trehalose, sorbitol, mannitol, glycerol, lactose, sorbitol,dextrose, raffinose, polyethylene glycol, hydroxyethyl starch, glycineglutamic acid, alanine, dextran, PVP (poly vinyl pyrrolidone), or anycombination thereof.

50. The pharmaceutical composition of item 49, wherein the tonicitymodifier is sodium chloride.

51. The pharmaceutical composition of item 48, 49 or 50, wherein thetonicity modifier is present in a concentration of about 30 mM to about250 mM.

52. The pharmaceutical composition of any one of items 38 to 51, whichfurther comprises a bulking agent.

53. The pharmaceutical composition of item 52, wherein the bulking agentis glycine, glutamic acid, alanine, mannitol, sorbitol, hydroxyethylstarch, dextran, PVP (poly vinyl pyrrolidone), mannitol, polyethyleneglycol, sorbitol, maltitol, lactitol, maltotriitol, xylitol or anycombination thereof.

54. The pharmaceutical composition of item 53, wherein the bulking agentis mannitol or sorbitol.

55. The pharmaceutical composition of item 52, 53 or 54, wherein thebulking agent is in a concentration of about 50 mM to about 300 mM.

56. The pharmaceutical composition of any one of items 38 to 55, whereinthe pharmaceutical composition has an osmolality between about 180 mOsmand about 350 mOsm.

57. The pharmaceutical composition of any one of items 38 to 61, whereinthe pharmaceutical composition has a pH of about 6.5 to about 8.0.

58. The pharmaceutical composition of any one of items 38 to 57, whereinsaid plasminogen or a biologically active variant thereof is humanplasminogen.

59. The pharmaceutical composition of any one of items 38 to 58, whereinsaid plasminogen or a biologically active variant, thereof consists ofmore than 80% of Glu-plasminogen.

60. The pharmaceutical composition of any one of items 38 to 58, whereinsaid plasminogen or a biologically active variant thereof isGlu-plasminogen.

61. The pharmaceutical composition of any one of items 38 to 60, for useas a medicament.

Further aspects of the invention will be apparent to a person skilled inthe art from the following description, claims, and generalizationsherein.

BRIEF DESCRIPTION OF THE FIGURES

FIG. 1A is a schematic of the primary structure of plasminogen showingits kringle domains, plasmin cleavage site and streptokinase bindingsite.

FIG. 1B shows the amino acid sequence of human plasminogen precursor.The sequence of the mature form is in bold, and the two lysine residuesbetween which there is a cleavage to generate Lys-Pg are underlined.

FIG. 2 is a picture of a vial containing the placebo (without Pg) ofFormulation 1 after lyophilisation.

FIG. 3 is a picture a vial containing Formulation 1 (described in Table1A) after lyophilisation.

FIG. 4 is a picture a vial containing the placebo (without Pg) ofFormulation 2 after lyophilisation.

FIG. 5 is a picture a vial containing Formulation 2 (described in Table1A) after lyophilisation.

FIG. 6 is a picture a vial containing the placebo (without Pg) ofFormulation 3 after lyophilisation.

FIG. 7 is a picture a vial containing Formulation 3 (described in Table1A) after lyophilisation.

FIG. 8 is a picture a vial containing the placebo (without Pg) ofFormulation 4 after lyophilisation.

FIG. 9 is a picture a vial containing Formulation 4 (described in Table1A) after lyophilisation.

FIG. 10 is a picture a vial containing the placebo (without Pg) ofFormulation 5 after lyophilisation.

FIG. 11 is a picture a vial containing Formulation 5 (described in Table1A) after lyophilisation.

FIG. 12 a picture of vials of lyophilized composition no. F0498 whichcontains plasminogen 20 mg/ml in 10 mM Tris-HCl, 35 mM NaCl, pH 8.0, and28.5 mM (0.5%) Arginine.

FIG. 13 a picture of vials of lyophilized composition no. F0449 whichcontains plasminogen 10 mg/ml in 10 mM Na Phosphate, 35 mM NaCl, pH 7.2,28.5 mM (0.5%) Arginine, and 54 mM (1%) Mannitol.

FIG. 14 a picture of vials of lyophilized composition no. F0459 whichcontains plasminogen 20 mg/ml in 10 mM Na Phosphate, 35 mM NaCl, pH 7.2,28.5 mM (0.5%) Arginine, and 54 mM (1%) Mannitol.

FIG. 15 is a picture of vials containing 5 mg/ml plasminogen in water(Sample 0), in 10 mM sodium citrate buffer pH 6.5 (Sample 1), 10 mMsodium phosphate buffer pH 7.2 (Sample 2) and 10 mM Tris-HCl buffer Ph8.0 (Sample 3) beside turbidity standards of 0.02, 20, 100, 800 NTUrespectively.

FIG. 16 is a picture of the same vials shown in FIG. 12, with theexception that 35 mM NaCl and 28.5 mM (0.5%) Arg have been added toSamples 1, 2 and 3.

FIGS. 17, 18 and 19 are graphs showing the turbidity over a period of 4hours for compositions containing 10 mg plasminogen, 35 mM NaCl with0.5% glycine, arginine, alanine and without amino acid, at pH 6.5 insodium citrate buffer (FIG. 17), pH 7.2 in sodium phosphate buffer (FIG.18), and at pH 8.0 in Tris buffer (FIG. 19).

FIGS. 20, 21 and 22 are graphs showing the turbidity over a period of 4hours for compositions containing 10 mg plasminogen, 35 mM NaCl, 0.5%mannitol with 0.5% glycine, arginine, alanine and without amino acid, atpH 6.5 in sodium citrate buffer (FIG. 20), pH 7.2 in sodium phosphatebuffer (FIG. 21), and at pH 8.0 in Tris buffer (FIG. 22).

DESCRIPTION OF SEQUENCES

SEQ ID NO:1 is a peptide useful according to the subject invention.

DETAILED DESCRIPTION OF THE INVENTION

Disclosed herein is a pharmaceutical composition comprising plasminogen,or a biologically active variant thereof.

Also disclosed herein is a pharmaceutical composition comprising:

-   -   plasminogen, or a biologically active variant thereof;    -   a tonicity modifier; and    -   a stabilising agent;

the composition having a pH of about 3.0 to about 10.0; preferably ofabout 5.0 to about 8.0; more preferably of about 6.0 to about 8.0; andfurther preferably of about 6.5 to about 7.5.

The present invention concerns a pharmaceutical composition comprisingplasminogen or a biologically active variant thereof, with an increasedrobustness. The present invention concerns a pharmaceutical compositioncomprising plasminogen or a biologically active variant thereof, thathas count of particles of 10 μm or greater that is lower than 6000particles per 100 mL of composition, or lower than 5000 particles per100 mL of composition, or lower than 4000 particles per 100 mL ofcomposition, or lower than 3000 particles per 100 mL of composition, orlower than 2000 particles per 100 mL of composition or lower than 1000particles per 100 mL of composition. In an embodiment of the presentinvention, the count of particles remains low after lyophilisation andreconstitution. In an embodiment of the present invention, repeatedpreparations of the same composition provide no variation of the countof particles.

The term “Plasminogen” as used herein refers to any form of a nativeplasminogen polypeptide (e.g., Glu-plasminogen or Lys-plasminogen) fromany animal, for example a mammal (e.g., human). Plasminogen is apro-enzyme is converted to the active enzyme, plasmin, by tissue-typeplasminogen activator (t-PA) or by urokinase plasminogen activator(u-PA). Plasmin then degrades fibrin and converts latent matrixmetalloproteinases (pro-MMPs) into active MMPs, which in turn furtherdegrade extracellular matrix (ECM) as part of the tissuehealing/remodeling process. The term “biologically active variant” asused herein refers to a mutated plasminogen polypeptide that retains thebiological activity of native plasminogen, i.e. the ability to beconverted to a plasmin polypeptide (by t-PA and or u-PA) that is able todegrade fibrin and converts latent matrix metalloproteinases (pro-MMPs)into active MMPs. The variant may comprises one or more amino acidsubstitutions, deletions/truncations (N-terminal, C-terminal, and/orinternal amino acid deletions/truncations), additions (N-terminal,C-terminal, and/or internal amino acid additions). The biologicallyactive variants may exhibit a biological activity (e.g., enzymaticactivity of the resulting plasmin) that is lower, higher or similar tothat of a native plasminogen polypeptide. In embodiments, the varianthas at least 60, 70, 75, 80, 85, 90, or 95% amino acid sequence identitywith a native plasminogen polypeptide. Biologically active plasminogenvariants are described, for example, in WO2012/093132, WO2013/024074 andin Wang et al. (1995, Protein Science 4, 1758-1767), and included thetruncated variants of plasminogen commonly referred to as“midiplasminogen”, “miniplasminogen”, “microplasminogen” and“delta-plasminogen” that lack one or more kringle domains and/or partsthereof. In an embodiment, the plasminogen is human plasminogen. In afurther embodiment, the composition comprises native human plasminogen.The plasminogen may be obtained from several sources. It may be obtainedby recombinant synthesis, or extracted/purified from blood, plasma or ablood-derived solution. Plasminogen can be extracted from blood orplasma by Cohn fractionation or by precipitation. Plasminogen can bepurified from plasma or blood-derived solution by a binding affinitychromatography, such as the method described in WO 2006/120423, orproduced recombinantly.

In an embodiment, the plasminogen or biologically active variant thereofis present in a concentration of about 0.01 mg/ml to about 80 mg/ml;preferably about 1 mg/ml to about 60 mg/ml; preferably about 5 mg/ml toabout 60 mg/ml; preferably about 5 mg/ml to about 40 mg/ml; preferablyof about 2 mg/ml to about 30 mg/ml, more preferably of about 2 mg/ml toabout 20 mg/ml, and further preferably of about 80, 70, 60, 50, 40, 30,25, 20, 15, 14, 13, 12, 11, 10, 9, 8, 7, 6, 5, 4, 3, 2, 1, 0.5, 0.1,0.05 or 0.01 mg/ml.

In an embodiment of the present invention, the plasminogen orbiologically active variant thereof contained in the pharmaceuticalcomposition has a purity of more than about 80%, or more than about 90%,or more than about 95%, or more than about 98%. The term “about” refersa variation of value by more or less 10%.

The pH of the present composition can be from about 3.0 to about 10.0;from about 5.0 to about 8.0; from about 6.0 to about 8.0; or from about6.5 to about 7.5. In embodiments, the pH of the present composition isabout 3.0, 3.5, 4.0, 4.5, 5.0, 5.5, 6.0, 6.1, 6.2, 6.3, 6.4, 6.5, 6.6,6.7, 6.8, 6.9, 7.0, 7.1, 7.2, 7.3, 7.4, 7.5, 8.0, 8.5, 9.0 or 10.0. Tomaintain the composition at such a pH, the present compositionpreferably comprises a buffer. Many buffers can be used within the scopeof the present invention, including the following buffers depending onthe desired pH:

Buffer Desired pH Citrate/phosphate 3.0, 3.5, 4.0, 4.5, 5.0, 5.5, 6.0,6.5 to 7.0, 7.5, 8.0, and any pH from Citrate 3.0 to 6.5 Acetate any pHfrom 3.6 to 5.6, for example 4.0, 4.5, 5.0, or 5.5 Sodium acetate 3.6 to5.6 Formate 3.0 to 4.5 Sodium formate any pH from 5.0 and 6.0, forexample 5.0, 5.5, or 6.0 Malate 4.0 to 6.0 Phosphate any pH from 6.5 and8.0, for example 6.5, 7.0, 7.5, or 8.0 PBS, TBS, TNT, PBT 7.0 to 7.5Sorensen's 5.8 to 8.0 Succinate any pH from 5.5 to 6.5, for example 6.0Carbonate any pH from 6.0 to 8.0, for example 6.5 Histidine any pH from5.0 and 7.0, for example 5.0, 5.5, 6.0, or 6.5 to 7.0 Borate 9 to 10Glycine-NaOH 8.6 to 10 Succinate 5.5 to 6.5 Maleate 5.5 to 7.2 Tris 7.2to 9.0 BIS-Tris any pH from 5.8 and 7.2 PIPES any pH from 6.1 and 7.5MOPS any pH from 6.2 and 7.6 HEPES any pH from 6.8 and 8.2 MES any pHfrom 5.5 and 6.7 ACES any pH from 6.1 and 7.5

In an embodiment, the buffer comprises a combination of two buffers;such as Citrate/Phosphate, Citrate/Histidine, Acetate/Histidine orSuccinate/Histidine. In an embodiment of the present invention, thebuffer does not comprise a citrate buffer. In another embodiment, thebuffer is citrate, phosphate (K₂HPO₄/NaHPO₄), histidine, or succinate.In another embodiment, the buffer is phosphate (K₂HPO₄/NaHPO₄),histidine, or succinate.

In an embodiment, the concentration of the buffer is from about 10 mM toabout 50 mM, or from about 10 mM to about 30 mM, for example about 2 mMor about 10 mM.

As used herein, a “tonicity modifier” refers to a compound which is usedto adjust the tonicity of the pharmaceutical composition. In anembodiment, the tonicity modifier is present in an amount that rendersthe composition isotonic. In another embodiment, the tonicity modifieris present in an amount that renders the composition hypertonic orhypotonic. In an embodiment, the tonicity modifier is sodium chloride,calcium chloride, magnesium chloride, potassium chloride, Na₂SO₄, ZnCl₂,borate, pharmaceutically acceptable monovalent salt, pharmaceuticallyacceptable divalent salt, pharmaceutically acceptable trivalent salt,pharmaceutically acceptable quatravalent salt, sucrose, trehalose,sorbitol, mannitol, glycerol, lactose, sorbitol, dextrose, cyclodextrin,raffinose, a sugar alcohol, polyethylene glycol, hydroxyethyl starch,glycine glutamic acid, alanine, dextran PVP (poly vinyl pyrrolidone), orany combination thereof. “Isotonic” is meant that the composition of thepresent invention has essentially the same osmolality than theosmolality of human blood. Isotonic compositions generally have anosmolality from about 250 to about 330 mOsm. Osmolarity can be measuredusing a vapor pressure or ice-freezing type osmometer, for example. Inan embodiment, the pharmaceutical composition comprises a mixture of twotonicity modifiers. In another embodiment, the pharmaceuticalcomposition comprises a mixture of three tonicity modifiers. In anembodiment, the tonicity modifier is a sugar or comprises a sugar, or amixture of sugars. In an embodiment, the tonicity modifier is sodiumchloride. In an embodiment, the concentration of the tonicity modifierconcentration is from about 30 mM to about 250 mM, or from about 20 mMto about 150 mM, or from about 30 mM to about 100 mM. In an embodiment,the concentration of the tonicity modifier concentration is about 34 mM,or about 35 mM, or about 68 mM, or about 75 mM. In an embodiment, theconcentration of the tonicity modifier is adjusted so that the resultingosmolality of the composition falls within the desired range.

In an embodiment, the composition further comprises a bulking agent. Asused herein, a “bulking agent” refers to a compound/agent whichincreases the bulk mass of a composition. Examples of bulking agentsinclude, without limitation, glycine, glutamic acid, alanine, mannitol,hydroethyl starch, dextran, PVP (poly vinyl pyrrolidone), mannitol,sorbitol, oligosaccharide-derived sugar alcohol, maltitol, lactitol,maltotriitol, xylitol, polyethylene glycol, sucrose, glucose, maltose,xorbitol, NaCl, or any combination thereof. These agents can also serveas tonicity modifiers. In embodiments, the concentration of bulkingagent is from about 50 mM to about 300 mM; or from about 100 mM to about200 mM, or about 150 mM, or from about 50 mM to about 150 mM, or about108 mM, or about 54 mM.

In an embodiment, the tonicity modifier and the bulking agent are inconcentrations such that the tonicity modifier/bulking agent ratio isabout 1.0 to about 15.0 (wt/wt); from about 1.0 to about 10.0, about 3.0to about 10.0, about 5.0 to about 10.0, for example about 5.0, about8.0, or about 10.0. In an embodiment, the concentration of bulking agentis adjusted based on the concentration of the tonicity modifier so thatthe tonicity modifier/bulking agent ratio is in the desired range.

In embodiments, the composition further comprises one or more additionalpharmaceutically acceptable ingredients such as carriers, excipients,diluents, stabilizers, buffers and the like.

In an embodiment, the pharmaceutical composition further comprises asugar, for example a reducing and/or a non-reducing sugar. As usedherein, a “reducing sugar” is one which contains a hemiacetal group thatcan reduce metal ions or react covalently with lysine and other aminogroups in proteins and a “non-reducing sugar” is one which does not havethese properties of a reducing sugar. In an embodiment, the reducingsugar is fructose, mannose, maltose, lactose, arabinose, xylose, ribose,rhamnose, galactose, glucose or any combination thereof. In anembodiment, the non-reducing sugar is sucrose, trehalose, sorbose,melezitose, raffinose or any combination thereof. In an embodiment, thepharmaceutical composition of the present invention is lyophilized inthe presence of a reducing sugar. In an embodiment, the pharmaceuticalcomposition of the present invention is lyophilized in the presence of anon-reducing sugar.

In embodiments, the pharmaceutical composition of the present inventionhas an osmolality of about 180 mOsm to about 350 mOsm; or of about 250mOsm to about 350 mOsm, or of about 200 mOsm to about 300 mOsm. Incertain embodiments, for example when the present composition isprepared for lyophilisation, it may have an osmolality higher than 350mOsm. In such a case, the reconstituted solution may have a volumesuperior than the volume of the composition in order to dilute thecomponents and reduce the osmolality. Also, when the composition isdesigned for the administration of a small volume, the osmolality of thecomposition may be higher than 350 mOsm.

As used herein, a “stabilising agent” refers to an agent that stabilisesplasminogen in solution. In an embodiment, the stabilising agentcomprises an amino acid, an amino acid salt, an amino acid analog or anymixture thereof. In a further embodiment, the amino acid, amino acidsalt, amino acid analog or mixture thereof is arginine, proline,glutamic acid, aspartic acid, glycine, alanine, cysteine, lysine, alysine analog such as epsilon-amino caproic acid, or any combinationthereof. In a preferred embodiment, said amino acid can be in the formof an amino acid base such as arginine hydrochloride and lysinehydrochloride. In an embodiment, the pharmaceutical compositioncomprises two amino acids, amino acid salts and/or amino acid analogs.In another embodiment, the pharmaceutical composition comprises threeamino acids, amino acid salts and/or amino acid analogs. In embodiments,the concentration of amino acid is from about 10 mM to about 300 mM,from about 20 mM to about 200 mM, or from about 10 mM to about 100 mM,or from about 25 mM to about 75 mM, or about 10 mM, or about 25 mM, orabout 50 mM, or about 75 mM, or about 100 mM, about 150 mM, about 200mM, about 250 mM, or about 300 mM.

In another embodiment, the pharmaceutical composition of the presentinvention further comprises an antioxidant. In an embodiment, theantioxidant is methionine, oxidized glutathione (Glu-Cys-Gly)₂ (613kDa), ascorbic acid, N-acetyl-L-cysteine/homocysteine, glutathione,6-hydroxy-2,5,7,8-tetramethylchroman-2-carboxylic acid (Trolox®), lipoicacid, sodium thiosulphate, platinum, glycine-glycine-histidine(tripeptide), butylated hydroxyltoluene (BHT), or any combinationthereof. In embodiments, the concentration of the antioxidant is fromabout 10 mM to about 200 mM, e.g., about 10 mM, about 50 mM, about 100mM, about 150 mM, or about 200 mM.

In another embodiment, the pharmaceutical composition of the presentinvention further comprises a surfactant. As used herein, a “surfactant”refers to a compound/agent that reduces interfacial tension between aliquid and a solid when dissolved in solution. In an embodiment, thesurfactant is Polysorbate® 80, Polysorbate® 20, Pluronic® F-68, or Brij®35; poloxamers (e.g. poloxamer 188 or Pluronic® F-68); Triton®; sodiumdodecyl sulfate (SDS); sodium laurel sulfate; sodium octyl glycoside;lauryl-, myristyl-, linoleyl-, or stearyl-sulfobetaine; lauryl-,myristyl-, linoleyl- or stearyl-sarcosine; linoleyl-, myristyl-, orcetyl-betaine; lauroamidopropyl-, cocamidopropyl-, linoleamidopropyl-,myristamidopropyl-, palmidopropyl-, or isostearamidopropyl-betaine (e.g.lauroamidopropyl); myristamidopropyl-, palmidopropyl-, orisostearamidopropyl-dimethylamine; sodium methyl cocoyl-, or disodiummethyl oleyl-taurate; and the MONAQUAT™ series (Mona Industries, Inc.,Paterson, N.J.), polyethyl glycol, polypropyl glycol, or copolymers ofethylene and propylene glycol, or any combination thereof. In anembodiment, the concentration of surfactant is adjusted to reduceaggregation of the plasminogen, biologically active variant or fragmentthereof and/or minimizes the formation of particulates in thecomposition and/or reduces adsorption. In an embodiment, the surfactantis present in the composition in a concentration of about 0.001-1%(w/v), about 0.002% to about 0.02% (w/v), or about 0.002% to about0.006% (w/v). In some embodiments, the composition comprises asurfactant which is a poloxamer. In some embodiments, the compositioncomprises Pluronic® F68. In particular embodiments, the compositioncomprises from about 0.01% (w/v) to about 1% (w/v) of Pluronic® F68, forexample about 0.1% (w/v) of Pluronic® F68. In an embodiment, thecomposition is substantially free or free of nonionic surfactant, e.g.,the composition comprises less than about 0.02%, 0.01%, 0.006%, or0.004% (w/v) of nonionic surfactant.

In another embodiment, the pharmaceutical composition of the presentinvention further comprises a polymeric stabilizer. In an embodiment,the polymeric stabilizer is Heparin (6 to 30 kDa); Polyaminoacid (2 to100 kDa) such as Poly (Glu), Poly (Asp), and Poly (Glu, Phe);Carboxymethyl cellulose (10-800 cps); Cyclodextrin; Dextran Sulphate, orany combination thereof. In embodiments, the concentration of thepolymeric stabilizer is from about 0.001% to about 0.50%, or from about0.001% to about 0.25%.

In another embodiment, the pharmaceutical composition of the presentinvention further comprises a polyethylene glycol, e.g., PEG 200, PEG400, PEG 1000, PEG, 4000, PEG, 8000, PEG 10 000, or any combinationthereof. In an embodiment, the concentration of the polyethylene glycolis from about 0.5% to about 10% (w/w).

In an embodiment, the pharmaceutical composition of the presentinvention further comprises a preservative. As used herein, a“preservative” refers to a compound/agent which can be added to thepharmaceutical composition to essentially reduce bacterial activitytherein, thus facilitating the production of a multi-use pharmaceuticalcomposition, for example. Examples of preservatives include m-cresol,benzyl alcohol, methanol, ethanol, iso-propanol, butyl paraben, ethylparaben, methyl paraben, phenol, glycerol, xylitol, resorcinol,catechol, 2,6-dimethylcyclohexanol, 2-methyl-2,4-pentadiol, dextran,polyvinylpyrrolidone, 2-chlorophenol, benzethonium chloride, merthiolate(thimersosal), benzoic acid (propyl paraben) MW 180.2, benzoic acid MW122.12, benzalkonium chloride, chlorobutanol, sodium benzoate, sodiumpropionate, or cetylpyridinium chloride. The preservative is selectedfor compatibility with the buffer and other components of thepharmaceutical composition (i.e., the solution is clear). For example,when the buffer is sodium acetate or sodium phosphate, compatiblepreservatives include methanol, ethanol, iso-propanol, glycerol,resorcinol, 2-methyl-2,4-pentadiol, merthiolate (thimerosal),benzalkonium chloride, sodium benzoate, or cetylpyridinium chloride. Theconcentration of the preservative used in the present composition can bedetermined according to the judgment of those of skill in the art. Insome embodiments, the concentration of the preservative is about 0.005to about 10% (w/v), about 0.1 to about 1.0% (w/v), or about 0.3 to about0.7% (w/v). In some embodiments, the concentration of the preservativeis about 0.005, 0.1, 0.3, 0.5, 0.7, or 1.0% (w/v).

In another embodiment, the pharmaceutical composition of the presentinvention is free of preservative.

One or more additional pharmaceutically acceptable carriers, excipientsor stabilizers, such as those described in Remington's PharmaceuticalSciences 19^(th) edition, Genarro, A. Ed. (1995) can be included in thepresent composition provided that they do not significantly adverselyaffect the desired characteristics of the pharmaceutical composition ofthe invention. Additional constituent elements of the composition of thepresent invention can include water, e.g., water for injection,vegetable oil, a thickening agent such as methylcellulose, ananti-adsorbant, a wetting agent, antioxidants including ascorbic acidand methionine, chelating agents such as EDTA, metal complexes (e.g.Zn-protein complexes), biodegradable polymers such as polyesters, and/orsalt-forming counter-ions such as sodium etc. Acceptable carriers,excipients or stabilizers are present in an amount such that they arenontoxic to subjects at the dosages and concentrations employed.

In an embodiment, the pharmaceutical composition of the presentinvention has a total weight of solid of from about 3.5% to about 8%when lyophilized, preferably from about 3.5% to about 6%, and morepreferably of from about 3.8% to about 5.5%. Increasing theconcentration of components in the pharmaceutical composition of theinvention results in increasing the total % of solid in the lyophilizedcomposition and improving the cake appearance of the lyophilisedcomposition.

In an embodiment, plasminogen or a variant thereof is Lys-plasminogen orGlu-plasminogen. In another embodiment, plasminogen or a variant thereofis Glu-plasminogen. In an embodiment, plasminogen or a variant thereofis composed various types of plasminogen and/or variants. Preferably,the content of Glu-plasminogen represents more than 50% of the totalcontent of plasminogen or variants thereof, or more than 60%, or morethan 70%, or more than 80%, or more than 90%.

An embodiment of the present invention is a pharmaceutical compositioncomprising plasminogen or a variant thereof, wherein the total amount ofprotein other than the plasminogen or variant thereof in the compositionis less than about 10%, less than about 5%, or less than about 2%. Anembodiment of the present invention is a pharmaceutical compositioncomprising plasminogen or a variant thereof, wherein the compositiondoes not comprise or is substantially free of, an additional protein(i.e. in addition to the plasminogen or variant thereof). In anembodiment of the present invention, the composition does not compriseor is substantially free of albumin. In an embodiment of the presentinvention, the composition does not comprise or is substantially free ofaprotinin. In an embodiment of the present invention, the compositiondoes not comprise or is substantially free of a trypsin inhibitor. In anembodiment of the present invention, the composition does not compriseor is substantially free of a serine protease inhibitor. In anembodiment of the present invention, the composition does not compriseor is substantially free of plasmin. In an embodiment of the presentinvention, the composition is substantially free or free of asurfactant, i.e. the concentration of surfactant is less than 0.01 mM.

In an embodiment, the pharmaceutical composition comprising plasminogenof the present invention is stable at room temperature for at least 2hours, 24 hours, a week or a month.

In an embodiment, the pharmaceutical composition comprising plasminogenof the present invention is stable at temperature below 0° C. for atleast 3 months, for at least 6 months, for at least 12 months or for atleast 24 months. Said temperature below 0° C. is about −20° C., about−30° C., about −60° C., about −70° C. or about −80° C.

In embodiments, the pharmaceutical composition comprising plasminogen isa liquid composition, a liquid composition suitable for lyophilisation,a liquid composition suitable for freezing, a lyophilised composition, afrozen composition or a reconstituted composition.

The term “pharmaceutical composition” designates a composition forpharmaceutical, medical or therapeutic purposes.

All combinations of the components described herein (pH, buffer,tonicity modifier, bulking agent, stabilising agent, etc.) and theirrespective concentrations for the pharmaceutical composition of thepresent invention are specifically envisioned by the present disclosure.Representative examples of pharmaceutical compositions of plasminogen(Pg) according to the present invention are represented in Table 1A.

TABLE 1A Tonicity No. Pg Buffer Bulking agent Stabilising agent modifierpH 1 5 mg/ml 10 mM Citrate  73 mM Sucrose 67 mM Glycine  68.4 mM NaCl;6.5 2 5 mg/ml 10 mM Citrate  73 mM Sucrose 33.5 mM Glycine 34.22 mM NaCl6.5 3 5 mg/m1 10 mM Citrate 117 mM Sucrose 67 mM Glycine  68.4 mM NaCl;6.5 4 5 mg/ml 10 mM Citrate 117 mM Sucrose 67 mM Glycine 34.22 mM NaCl6.5 5 5 mg/ml 10 mM Citrate  73 mM Sucrose 67 mM,Glycine 34.22 mM NaCl6.5 6 5 mg/ml 10 mM Citrate  73 mM Sucrose 67 mM Glycine  75.3 mM NaCl6.5 7 5 mg/ml 10 mM Citrate  79 mM Sucrose 40 mM Glycine    75 mM NaCl6.5 8 5 mg/ml 10 mM Citrate  77 mM Sucrose 47 mM Glycine    75 mM NaCl6.5 9 5 mg/ml 10 mM Citrate  75 mM Sucrose 54 mM Glycine    75 mM NaCl6.5 10 5 mg/ml 10 mM Citrate 50-150 mM 25 to 75 mM 35-150 mM 6.5 SucroseGlycine NaCl 11 5 mg/ml 10 mM Citrate 50-150 mM 25 to 75 mM 35-150 mM6.5 Mannitol Glycine NaCl 12 5 mg/ml 10 mM Citrate 50-150 mM 25 to 75 mM35-150 mM 6.5 Sorbitol Glycine NaCl 13 5 mg/ml 10 mM Citrate 50-150 mM25 to 75 mM 35-150 mM 6.5 Sucrose Arginine NaCl 14 5 mg/ml 10 mM Citrate50-150 mM 25 to 75 mM 35-150 mM 6.5 Mannitol Arginine NaCl 15 5 mg/ml 10mM Citrate 50-150 mM 25 to 75 mM 35-150 mM 6.5 Sorbitol Arginine NaCl 165 mg/ml 10 mM 50-150 mM 25 to 75 mM 35-150 mM 7.2 Phosphate SucroseGlycine NaCl 17 5 mg/ml 10 mM 50-150 mM 25 to 75 mM 35-150 mM 7.2Phosphate Mannitol Glycine NaCl 18 5 mg/ml 10 mM 50-150 mM 25 to 75 mM35-150 mM 7.2 Phosphate Sorbitol Glycine NaCl 19 5 mg/ml 10 mM 50-150 mM25 to 75 mM 35-150 mM 7.2 Phosphate Sucrose Arginine NaCl 20 5 mg/ml 10mM 50-150 mM 25 to 75 mM 35-150 mM 7.2 Phosphate Mannitol Arginine NaCl21 5 mg/ml 10 mM 50-150 mM 25 to 75 mM 35-150 mM 7.2 Phosphate SorbitolArginine NaCl

Other representative examples of pharmaceutical compositions accordingto the present invention are represented in Table 1B below forcompositions of about 0.01 mg/ml to about 80 mg/ml and preferably from 5to 60 mg/ml of plasminogen or variant thereof and having a pH from 6.5to 8.0. In all these examples, the content of plasminogen is preferablymainly composed of Glu-plasminogen, and the total content of plasminogenor variants is from 5 mg/mL to 60 mg/mL.

TABLE 1B No. Bulking agent Stabilising agent Tonicity modifier 1 — — 35mM NaCl 2 — 25 mM Arginine HCl 35 mM NaCl 3 — 50 mM Arginine HCl 35 mMNaCl 4  50 mM Mannitol 25 mM Arginine HCl 35 mM NaCl 5  50 mM Mannitol50 mM Arginine HCl 35 mM NaCl 6 100 mM Mannitol 25 mM Arginine HCl 35 mMNaCl 7 100 mM Mannitol 50 mM Arginine HCl 35 mM NaCl 8  50 mM Sorbitol25 mM Arginine HCl 35 mM NaCl 9  50 mM Sorbitol 50 mM Arginine HCl 35 mMNaCl 10 100 mM Sorbitol 25 mM Arginine HCl 35 mM NaCl 11 100 mM Sorbitol50 mM Arginine HCl 35 mM NaCl 12 — — 50 mM NaCl 13 — 25 mM Arginine HCl50 mM NaCl 14 — 50 mM Arginine HCl 50 mM NaCl 15  50 mM Mannitol 25 mMArginine HCl 50 mM NaCl 16  50 mM Mannitol 50 mM Arginine HCl 50 mM NaCl17 100 mM Mannitol 25 mM Arginine HCl 50 mM NaCl 18 100 mM Mannitol 50mM Arginine HCl 50 mM NaCl 19  50 mM Sorbitol 25 mM Arginine HCl 50 mMNaCl 20  50 mM Sorbitol 50 mM Arginine HCl 50 mM NaCl 21 100 mM Sorbitol25 mM Arginine HCl 50 mM NaCl 22 100 mM Sorbitol 50 mM Arginine HCl 50mM NaCl 23 — 25 mM Arginine HCl — 24 — 50 mM Arginine HCl — 25 — 75 mMArginine HCl — 26 — 100 mM Arginine HCl  — 27 — 25 mM Arginine HCl — 28— 50 mM Arginine HCl — 29 — 75 mM Arginine HCl — 30 — 100 mM ArginineHCl  —

In an embodiment, the pharmaceutical composition comprising plasminogenof the present invention is suitable for intravenous, subcutaneous,topical, intradermal, ophthalmic and/or intramuscular administration.

According to an embodiment, the pharmaceutical composition of thepresent invention is for administration in a human subject. Preferably,the pharmaceutical composition of the present invention is forintravenous, subcutaneous, topical, intramuscular or ophthalmicadministration. In an embodiment, the composition of the presentinvention is in the form of a liquid, gel, cream, or ointment.

The plasminogen that is formulated in the composition of the presentinvention may be obtained from several sources. It may be obtained byrecombinant synthesis, or extracted/purified from blood, plasma or ablood-derived solution. Plasminogen can be extracted from blood orplasma by Cohn fractionation or by precipitation. Plasminogen can bepurified from plasma or blood-derived solution by a binding affinitychromatography, such as the method described in WO 2006/120423. Avariant of plasminogen includes, without limitation, any modificationsof the amino acid sequence or any additions of a group thereto or anyadditions of an amino acid or an amino acid sequence thereto. A fragmentof plasminogen includes, without limitation, any deletions of an aminoacid or an amino acid sequence thereto.

As used herein, the term “about” intends to cover + or −10% of thecorresponding value.

The term “subject” includes living organisms which can benefit from anadministration of plasminogen, a variant thereof or a fragment thereof.The term “subject” includes animals such as mammals or birds.Preferably, the subject is a mammal. More preferably, the subject is ahuman. Most preferably, the subject is a human patient in need oftreatment.

As used herein, “preventing” or “prevention” is intended to refer to atleast the reduction of likelihood of the risk of (or susceptibility to)acquiring a disease or disorder (i.e., causing at least one of theclinical symptoms of the disease not to develop in a patient that may beexposed to or predisposed to the disease but does not yet experience ordisplay symptoms of the disease). Biological and physiologicalparameters for identifying such patients are provided herein and arealso well known by physicians.

The terms “treatment” or “treating” of a subject includes theapplication or administration of a composition of the invention to asubject (or application or administration of the composition of theinvention to a tissue or an organ of a subject) with the purpose ofdelaying, stabilizing, curing, healing, alleviating, relieving,altering, remedying, less worsening, ameliorating, improving, oraffecting the disease or condition, the symptom of the disease orcondition, or the risk of (or susceptibility to) the disease orcondition. The term “treating” refers to any indication of success inthe treatment or amelioration of an injury, pathology or condition,including any objective or subjective parameter such as abatement;remission; lessening of the rate of worsening; lessening severity of thedisease; stabilization, diminishing of symptoms or making the injury,pathology or condition more tolerable to the subject; slowing in therate of degeneration or decline; making the final point of degenerationless debilitating; or improving a subject's physical or mentalwell-being. In some embodiments, the term “treating” can includeincreasing a subject's life expectancy and/or delay before additionaltreatments are required.

As used herein, the term “therapeutically effective amount” means theamount of compound that, when administered to a subject for treating orpreventing a particular disorder, disease or condition, is sufficient toeffect such treatment or prevention of that disorder, disease orcondition. As used herein, the term “therapeutically effective amount”further means the amount of plasminogen, its variant or its fragmentthat is effective to heal a wound, heal a tympanic membrane perforation,heal a periodontal wound, treat an infectious disease, increase ormaintain oral health, treat posterior vitreous detachment (PVD),diabetic ulcer, and plasminogen-deficient subjects thrombolysisindications, such as coronary thrombosis, treating reperfusion injury totissue, treating ischemia, infarction, brain edema, for improving themicrocirculation, treat plasminogen-deficient subjects, and modulate ofcomplement pathway in subjects. Dosages and therapeutically effectiveamounts may vary for example, depending upon a variety of factorsincluding the activity of the age, body weight, general health, gender,and diet of the subject, the time of administration, the route ofadministration, the rate of excretion, and any drug combination, ifapplicable, the effect which the practitioner desires the compound tohave upon the subject, and the particular disorder(s) the subject issuffering from. In addition, the therapeutically effective amount maydepend on the subject's blood parameters (e.g., lipid profile, insulinlevels, glycemia), the severity of the disease state, organ function, orunderlying disease or complications. Such appropriate doses may bedetermined using any available assays including the assays describedherein. When the pharmaceutical composition of the invention is to beadministered to humans, a physician may for example, prescribe arelatively low dose at first, subsequently increasing the dose until anappropriate response is obtained. The dose to be administered willultimately be at the discretion of the physician.

Kits

The compound(s) of the invention may be packaged as part of a kit,optionally including a container (e.g., packaging, a box, a vial, etc.).The kit may be commercially used according to the methods describedherein and may include instructions for use in a method of theinvention.

The composition of the invention may or may not be administered to apatient at the same time than another active ingredient, before orafter, and by the same route of administration or by another route ofadministration. Therefore, the present invention also encompass kitswhich, when used by the medical practitioner, can simplify theadministration of appropriate amounts of the present composition aloneor in combination with another active ingredient to a patient.

Kits of the invention can further comprise a pharmaceutically acceptableliquid for reconstitution of a lyophilised composition. For example, ifan active ingredient is provided in a solid form that must bereconstituted for parenteral administration, the kit can comprise asealed container of a suitable pharmaceutically acceptable liquid inwhich the active ingredient can be dissolved to form a particulate-freesterile solution that is suitable for parenteral administration.

In certain embodiments, the present composition is contained in a vial,bottle, tube, syringe or other container for single or multipleadministrations. Such containers can be made of glass or a polymermaterial such as polypropylene, polyethylene, polyvinylchloride, orpolyolefin, for example. In some embodiments, the containers can includea seal, or other closure system, such as a rubber stopper that can bepenetrated by a needle in order to withdraw a single dose and thenre-seal upon removal of the needle. All such containers for injectableliquids, lyophilized compositions, reconstituted lyophilizedcompositions or powders for reconstitution and injection known in theart are contemplated for use in the presently disclosed compositions andmethods. In a particular embodiment, the container is a pen-typedelivery apparatus comprising a single dose or multiple doses. Such apen-type delivery apparatus can be permanent, e.g., a permanent pen thathouses a disposable cartridge containing a single dose or multipledoses, or the entire apparatus can be disposable, e.g., a disposable penthat contains a single dose or multiple doses. In certain embodimentswhere the pen-type delivery apparatus comprises multiple doses, the dosecan be pre-set, i.e., fixed. In other embodiments, the dose can be aflexible dose, i.e., dialed-in by the user. In some embodiments, thepen-type delivery apparatus comprises a luer-lock, luer-cone, or otherneedle fitting connector that facilitates attachment of a disposableneedle. In other embodiments, the pen-type delivery apparatus comprisesa staked, i.e., permanent needle. In another particular embodiment, thecontainer is a syringe. In some embodiments, the syringe comprises aluer-lock, luer-cone, or other needle fitting connector that facilitatesattachment of a disposable needle. In other embodiments, the syringecomprises a staked, i.e., permanent, needle. In some embodiments, thesyringe is prefilled with a single dose or multiple doses.

Stability Assays

“Stable” compositions include compositions in which the protein(plasminogen or variant thereof) therein essentially retains itsphysical stability and/or chemical stability and/or biological activityupon storage. Various analytical techniques for measuring proteinstability are available in the art and are reviewed in Lee, V., 1991,Peptide and Protein Drug Delivery, 247-301 (Marcel Dekker, Inc., NewYork, N.Y.) and Jones, A. 1993, Adv. Drug Delivery Rev. 10: 29-90, forexample. Stability can be measured at a selected temperature for aselected time period. In an embodiment, the composition is stable atroom temperature (about 25° C.) or at 40° C., for at least 1, 2, 3, 4, 5or 6 months and/or stable at about 2-8° C. for at least 1, 2, 3, 4, 5 or6 months. Furthermore, in certain embodiments, the composition is stablefollowing freezing (e.g., −30° C.). In certain embodiments, the criteriafor stability are as follows: (1) the composition remains clear byvisual analysis; (2) the concentration, pH and osmolality of thecomposition has no more than about ±10% change; (3) no more than about10%, no more than about 5%, or no more than about 1% of aggregate formsas measured by SEC-HPLC; and (4) no more than 10%, no more than about5%, or no more than 3% of plasminogen breaks down as measured by knownin the art and well established analytical methods.

Thermal Denaturation: The thermal denaturation experiments HTSformulation screening for Plasminogen or a variant thereof or a fragmentthereof can performed using Optim 2™ from Pall; or other alternativebiophysical technique(s) to monitor the unfolding of the protein(Standard PCR (with a heating block), Fluorimeter or CDspectrophotometer (with temperature control). As an example, the Optim 2system simultaneously measure a range of protein stability-indicatingparameters including unfolding transition temperature (Tm), aggregationonset temperature (Tagg) and rates of aggregation.

The effects of excipients on the thermostability: Narrow pH isdetermined at the optimum pH range with higher Melting Temperature (Tm)and Aggregation Onset Temperature (Tagg). If the higher Tm is obtainedin the physiological range of 6.5-7.5; the next test(s) is to screenwith at least 3 buffer system(s).

Additionally, IEF (isoelectric focusing) or cIEF (capillary isoelectricfocusing) can be tested for evaluation the physical conformation ofplasminogen in solution.

The percentage of stabilization based on plasminogen enzyme activitymeasurements, for each buffer/excipient and for a reference solutioncontaining only plasminogen after 1-8 hours of incubation at 60° C. Inanother test, the stabilization may be evaluated at 37° C. for 1-8hours. The percentage of stabilization is calculated as:

Percent of Stabilization=100×[S−T]/[T]

Where S is the residual enzyme activity of the enzyme solution with theexcipient to be tested after 10 days at 35° C.; and T is the activity ofthe reference without any excipient stored in the same conditions.

Aggregation and Particle Measurement Count (PMC): The PMC is calculatedby the Light Obscuration Particle Count Test. Analysts should use asuitable instrument that is based on the principle of light blockage andthat allows an automatic determination of particle size and number. Thesensor selected must be appropriate for the intended particle size rangeand anticipated particle count. Standards particle size range aregenerally between 2 and 100 mm. Analysts should verify the performanceof the apparatus using the USP Particle Count RS dispersed inparticle-free water at an appropriate volume. Care must be taken toavoid agglomeration of particles during dispersion in the calibrationprocess.

Methods: Product samples are tested in the manner that most suitablyrepresents delivery (e.g., expelled syringe contents). For parenteralsthat have a sufficient volume (i.e., volume large enough to facilitatetesting), testing of individual units is often diagnostic. Forparenteral products that do not have a sufficient volume, carefully andthoroughly mix each unit, then combine the contents of a suitable numberof units in a separate container to obtain the volume required for asingle test (generally 0.2-5.0 mL). If a dilution is performed, ensurethat the blank container has sufficient volume for product and diluentsand that few, if any, particles are introduced in the process. Open eachunit cautiously, remove the sealing closure, and avoid contamination ofthe contents before sampling, diluting, or, if necessary, pooling. Whena product solvent is specified, e.g., for lyophilized solids or powdersfor parenteral use, the reconstitution or dilution must be performedwith the appropriate amount of specified solvent. In this case, thesolvent itself may be tested to ensure that it is not a significantsource of particles. Subtraction of the solvent particle count from thetotal count is not allowed. Eliminating gas bubbles is a key step,especially for proteinaceous products that readily entrain gas. Twomethods are recommended: either allowing the product fluid to standunder ambient pressure or applying a gentle (e.g., 75 Torr) vacuum.Other methods may be used when demonstrated to be suitable. Sonicationshould be avoided. Once the samples have been degassed, they must beremixed gently to suspend all particles by mixing the contents of thesample gently but thoroughly by an appropriate means, e.g., slowswirling of the container by hand. Inversion to mix the product fluid isnot recommended at any time. Immediately after mixing, withdraw NLT fouraliquots, each of a volume appropriate for the instrument's capacity(generally 0.2-5.0 mL). Count the number of particles over the selectedsize range, including particles equal to or greater than 10 and 25 mm.Disregard the result obtained for the first aliquot, and calculate themean number of particles at each size range for the remaining aliquotsof the preparation being tested. A tare volume also can be applied tocontrol sampling that should be representative of the sensor dynamicrange and needle volume.

Evaluation: Regulatory authorities like the FDA (Food and DrugAdministration) and USP/EP/JP (United State Pharmacopoeia) Chapters(787, 788 and 789), EP European Pharmacopoeia (2.9.20 ParticulateContamination), and JP Japanese Pharmacopoeia JP (16: 6.06. 6.07 ForeignInsoluble Matter Test) requirements on Particulate Matter define theacceptable level of particles in liquid preparation for human use asfollow. For parenteral products that are therapeutic protein injectionsfor infusion or injection supplied in containers with a nominal contentof less than or equal to 100 mL: the average number of particles presentin the units tested should not exceed 6000 per container equal to orgreater than 10 μm and should not exceed 600 per container equal to orgreater than 25 μm. For therapeutic protein injections supplied incontainers with a nominal content of more than 100 mL, and parenteralinfusion preparations or injections with a nominal content of more than100 mL: the average number of particles present in the units testedshould not exceed 25 per mL equal to or greater than 10 μm and shouldnot exceed 3 per mL equal to or greater than 25 μm. Also, total particleload should not exceed 6000 per container equal to or greater than 10 μmand should not exceed 600 per container equal to or greater than 25 μm.Products that are used with a final filter during administration(in-line) are exempt from these requirements, providing that scientificdata are available to justify the exemption. However, filtrates areexpected to comply with the guideline. For products supplied or firstreconstituted in <100 mL, and then diluted for infusion in a volume>100mL, particle content should be assessed both before and after dilutionand evaluated based on their final volume. Hence, USP/EP/JP Method areherein included in this document. Herein, the term “Particle” refers to“Particulate” and vice-versa; these terms can be used intechangeably.

Kinetic Study Measured by light scattering at 476 nm/600 nm using theOptim-2™ or by Standard UV Spectrophotometer at UV at 350 nm:Accelerated conditions such as elevated temperature (65° C.) and acidicsolution pH (4.5) or basic solution pH (9.5) is selected to more rapidlyscreen for different stabilizing excipients. The components of thecomposition are evaluated to increase the Tagg as a function of time ata fixed temperature (55-60° C.) for at least 1 to 4 hours. Theaggregation kinetic behavior of the Plasminogen solution is measured at55-60° C. equipped with temperature control (onset Tm is determined) forup to 4 h. The excipients are screened for their effect on inhibition ofPlasminogen aggregation as measured by the light scattering at 267 nm,467 nm or 350 nm (Cheng, W. et al. “Comparison of High-ThroughputBiophysical Methods to Identify Stabilizing Excipients for a Model IgG2Monoclonal Antibody:Conformational Stability and Kinetic AggregationMeasurements” Journal of Pharmaceutical Sciences, Vol. 101, No 5, page1701-1720, 2012).

The effect of protein concentration of thermal stability: One of theadvantages of Optim-2™ or Dual Scanning Fluorimeter (DSF with the SyproDye) is the ability to carry out measurements of Tm and Tagg in highlyconcentrated protein solutions. Whether investigating potentialcandidates for relative stability, or screening different formulationconditions for stabilization of proteins, it may be important toidentify the properties of the protein solution at high concentrationswhere the number of characterization methods available is limited.

The effect of formulation composition on the final Osmolality: In thefinal pharmaceutical composition, the buffer, excipient and saltconcentrations are not preferred to be very high due to the practicallimitations of parental injection. Instead, more moderate concentrationssuch as more physiological are preferred in order to obtain anOsmolality in the range of 240 to 400 mOsm. Therefore, Osmolality iscalculated to initially determine this range; however for IV injection ahigher Osmolality concentrations can be also be used during thedevelopment of the freeze drying process if after reconstitutionPlasminogen or variant or fragment thereof is diluted by the solutionfor reconstitution.

It should be noted that the term “Formulation” and “Composition” may beused herein alternatively and are intended to designate the same.

Headings are included herein for reference and to aid in locatingcertain sections These headings are not intended to limit the scope ofthe concepts described therein, and these concepts may haveapplicability in other sections throughout the entire specificationThus, the present invention is not intended to be limited to theembodiments shown herein but is to be accorded the widest scopeconsistent with the principles and novel features disclosed herein.

The singular forms “a”, “an” and “the” include corresponding pluralreferences unless the context clearly dictates otherwise.

Unless otherwise indicated, all numbers expressing quantities ofingredients, reaction conditions, concentrations, properties, and soforth used in the specification and claims are to be understood as beingmodified in all instances by the term “about”. At the very least, eachnumerical parameter should at least be construed in light of the numberof reported significant digits and by applying ordinary roundingtechniques. Accordingly, unless indicated to the contrary, the numericalparameters set forth in the present specification and attached claimsare approximations that may vary depending upon the properties sought tobe obtained. Notwithstanding that the numerical ranges and parameterssetting forth the broad scope of the embodiments are approximations, thenumerical values set forth in the specific examples are reported asprecisely as possible. Any numerical value, however, inherently containcertain errors resulting from variations in experiments, testingmeasurements, statistical analyses and such.

It is understood that the examples and embodiments described herein arefor illustrative purposes only and that various modifications or changesin light thereof will be suggested to persons skilled in the art and areto be included within the present invention and scope of the appendedclaims.

EXAMPLES

The following examples further illustrate the practice of this inventionbut are not intended to be limiting thereof.

Example 1: Stability of Plasminogen in a Frozen Formulation

Stability testing results for a pharmaceutical composition comprising 5mg/ml of human plasminogen extracted from plasma in accordance with abinding affinity technique described in WO 2006/120423, 10 mM Citrate,150 mM NaCl, at pH 6.5 (Lot: 2002.pc02_Pg140210.01) were stored at −20°C., −30° C. and −80° C. for up to 6 Months. The stability data indicatedthat plasminogen is stable under these frozen conditions. Plasminogenlot: 2002.pc02_Pg140210.01 was tested at −20° C., −30° C. and −80° C.The level of aggregation was investigated by SEC-HPLC, the level ofpurity by SDS-PAGE under reducing, and also under non-reducingconditions, the level of activity by BCS activity and the proteinconcentration by absorbance at 280 nm (ABS₂₈₀). The results of thestability data after 6 months demonstrated that all the stabilityindicating assays achieved the desired specifications.

Example 2: Osmolality Analysis and Total Percentage of Solid Analysis

The osmolality of formulations 1 to 6 and the total percentage of solidafter lyophilisation for formulations 1-9 are reported in Table 2. Thecontent of Formulations 1-9 is defined in Table 1A.

TABLE 2 Total % of Solid Formulation Plasminogen Sucrose Glycine NaClCake Osmolality # mg/mL mM mM mM % mOsm 1 5 73 67 68 3.9 265 2 5 73 3434 3.4 198 3 5 117 67 68 5.4 309 4 5 117 67 34 5.2 275 5 5 73 67 34 3.7231 6 5 73 67 75 3.9 272 7 5 27 40 75 3,9 TBD 8 5 26.5 47 75 3,9 TBD 9 526 54 75 3.9 TBD

The osmolality is in the physiological range of 240-400 mOsm. Theresulting osmolality of the tested compositions are advantageouslyfalling within or very close to the physiological range of osmolality.The total % of solid cake (g of solid composition after lyophilisationper 100 ml of the liquid composition before lyophilization) is around 4%for all compositions and placebos, which is within the preferred rangeof 3.5 to 8%.

Example 3: Molecular Weight of Plasminogen

The molecular weight of the plasminogen purified and formulated in thepharmaceutical composition of the present invention has a molecularweight of 87,000 (MW), determined by calculation of the primary sequenceof amino acids of the plasminogen and verified by mass spectrometry.

Example 4: Study of Appearance of the Cake after Lyophilisation

Formulations 1 to 5 have been lyophilised following the cycle processdetailed in Table 5. The content of Formulations 1-5 is defined in Table1A.

TABLE 5 Shelf Temperature Ramp Set Point, Time, Soak Time, Pressure SetStep [° C.] [minutes] [minutes] Point, [mT] Product Loading 5 — 60Atmospheric Freezing −50 120 240 Atmospheric Evacuation −50 — — 50Primary −22 90 3860 50 Secondary 35 120 270 50

FIGS. 2 to 11 are pictures of the vials containing 12.5 ml ofFormulations 1 to 5 respectively, or their corresponding placebo(without plasminogen). Thus, the effect of the presence of plasminogenon the stability of the composition has been investigated. The physicalinspection of the appearance of the cake that has been formed by thelyophilisation process. All cakes have maintained a uniform density,without any discoloration. Less than 10% of the tested vials for theplacebo of Formulations 2, 3 and 5 have provided a melt back. Less than10% of the tested vials for the placebo of Formulation 1 have collapsed.Less than 10% of the tested vials for the Formulations 1-5 and theplacebo of Formulation 4 have shown a basal retraction. Overall theappearance of the cake is satisfying.

The content of the composition dictates collapse temperature. Each pureamorphous excipient has a characteristic Tg′ and collapse temperature;the collapse temperature for the formulation is the mass averagedtemperatures of all of the compositions in the amorphous phase. It isimportant to design a formulation with maximum collapse temperature,because the rate of drying is directly proportional to the sampletemperature during lyophilization.

Finally, collapse temperature will be decreased if salts (NaCl) andexcipients are not maximally crystallized. For example, glycine has aTg′ of −45° C., and its contribution to the amorphous phase can reducecollapse temperature to impractically low values.

Example 5: Effect of Amino Acid on Plasminogen Aggregation in Solution

Plasminogen alone in water generates a number of plasminogen aggregatesthat is inacceptable by the FDA i.e. higher than 6000 particles of 10 μmor greater per 100 mL of composition (see Sample F0406 in Table 6).Various sucrose-based formulations have been developed to formulateplasminogen. Although most of these formulations succeed to meet thethreshold of 6000 particles of 10 μm or greater per 100 mL, formulationscontaining a lower particle content have been developed and the effectof amino acid to stabilise plasminogen is studied herein. These furtherformulations contain 10 mg of plasminogen and 35 mM NaCl, and treebuffers/pH have been tested, i.e. 10 mM Sodium Citrate (pH 6.5), 10 mMSodium Phosphate (pH 7.2) and 10 mM Tris HCL (pH 8.0). Have beencompared the presence and absence of an amino acid selected fromarginine, alanine and glycine at a concentration of 0.5%. The testedformulations are fresh, i.e. before lyophilisation. Table 6 reports thePMC counts for the particles of 10 μm or greater and of 25 μm orgreater. The presence of amino acid have shown a stabilizing effect hascontributed to reduce the aggregation level.

TABLE 6 Assigned Pre-Lyo Sample PMC Count/mL ID Sample Description 10 μm25 μm F0406 Pg Bulk in water at 10 mg/ml 1,039,061 20,181 Rx1 Pg (5mg/mL), 67 mM glycine 5,793 155 Rx5 Pg (5 mg/mL), 12 mM citrate buffer,73 mM sucrose, 68 mM NaCl, 5,495 243 67 mM glycine, pH 6.5 Rx6 Pg (5mg/mL), 12 mM citrate buffer, 117 mM sucrose, 68 mM NaCl, 4,068 94 pH6.5 Rx7 Pg (5 mg/mL), 12 mM citrate buffer, 117 mM sucrose, 68 mM NaCl,3,349 96 pH 6.5 F0080 Pg (10 mg/mL), 10 mM Sodium Citrate, 35 mM NaCl,pH 6.5 147 0 F0081 Pg (10 mg/mL), 10 mM Sodium Citrate, 35 mM NaCl, 1090 66 mM (0.5%) Glycine, pH 6.5 F0082 Pg (10 mg/mL), 10 mM SodiumCitrate, 35 mM NaCl, 122 1 28.5 mM (0.5%) Arginine, pH 6.5 F0083 Pg (10mg/mL), 10 mM Sodium Citrate, 35 mM NaCl, 91 1 26 mM (0.5%) Alanine, pH6.5 F0102 Pg (10 mg/mL), 10 mM Sodium Phosphate, 35 mM NaCl, pH 7.2 27118 F0103 Pg (10 mg/mL), 10 mM Sodium Phosphate, 35 mM NaCl, 124 6 66 mM(0.5%) Glycine, pH 7.2 F0104 Pg (10 mg/mL), 10 mM Sodium Phosphate, 35mM NaCl, 128 3 28.5 mM (0.5%) Arginine, pH 7.2 F0105 Pg (10 mg/mL), 10mM Sodium Phosphate, 35 mM NaCl, 180 2 26 mM (0.5%) Alanine, pH 7.2F0106 Pg (10 mg/mL), 10 mM Tris, 35 mM NaCl, pH 8.0 118 5 F0107 Pg (10mg/mL), 10 mM Tris, 35 mM NaCl, 66 mM (0.5%) Glycine, 91 7 pH 8.0 F0108Pg (10 mg/mL), 10 mM Tris, 35 mM NaCl, 28.5 mM (0.5%) Arginine, 75 3 pH8.0 F0109 Pg (10 mg/mL), 10 mM Tris, 35 mM NaCl, 26 mM (0.5%) Alanine,46 1 pH 8.0

Example 6: Effect of Mannitol Combined with Amino Acid on PlasminogenAggregation

The presence of the bulking agent, 0.5% mannitol, on the formulationscontaining 10 mg of plasminogen and 35 mM NaCl, with either 10 mM SodiumCitrate (pH 6.5), 10 mM Sodium Phosphate (pH 7.2) or 10 mM Tris HCL (pH8.0), with and without amino acid (0.5% arginine hydrochloride, alanineor glycine) have been tested and reported in Table 7. Fresh formulations(before lyo) were tested in this study. Generally, the presence ofmannitol has lowered the particle counts. Generally, the combination ofmannitol and amino acid contributes to lower the particle counts, andespecially with the combination of arginine with mannitol.

TABLE 7 Assigned Pre-Lyo Sample PMC Count/mL ID Sample Description 10 μm25 μm F0114 Pg (9 mg/mL), 10 mM Sodium Citrate, 35 mM NaCl, pH 6.5 3,61061 F0115 Pg (9 mg/mL), 10 mM Sodium Citrate, 35 mM NaCl, 805 8 27 mM(0.5%) Mannitol, pH 6.5 F0116 Pg (9 mg/mL), 10 mM Sodium Citrate, 35 mMNaCl, 962 13 66 mM (0.5%) Glycine, 27 mM (0.5%) Mannitol, pH 6.5 F0117Pg (9 mg/mL), 10 mM Sodium Citrate, 35 mM NaCl, 520 11 28.5 mM (0.5%)Arginine, 27 mM (0.5%) Mannitol, pH 6.5 F0118 Pg (9 mg/mL), 10 mM SodiumCitrate, 35 mM NaCl, 990 1 26 mM (0.5%) Alanine, 27 mM (0.5%) Mannitol,pH 6.5 F0119 Pg (9 mg/mL), 10 mM Sodium Phosphate, 35 mM NaCl, pH 7.2133 6 F0120 Pg (9 mg/mL), 10 mM Sodium Phosphate, 35 mM NaCl, 107 3 27mM (0.5%) Mannitol, pH 7.2 F0121 Pg (9 mg/mL), 10 mM Sodium Phosphate,35 mM NaCl, 191 6 66 mM (0.5%) Glycine, 27 mM (0.5%) Mannitol, pH 7.2F0122 Pg (9 mg/mL), 10 mM Sodium Phosphate, 35 mM NaCl, 95 2 28.5 mM(0.5%) Arginine, 27 mM (0.5%) Mannitol, pH 7.2 F0123 Pg (9 mg/mL), 10 mMSodium Phosphate, 35 mM NaCl, 113 5 26 mM (0.5%) Alanine, 27 mM (0.5%)Mannitol, pH 7.2 F0124 Pg (9 mg/mL), 10 mM Tris, 35 mM NaCl, pH 8.0 86 2F0125 Pg (9 mg/mL), 10 mM Tris, 35 mM NaCl, 578 10 27 mM (0.5%)Mannitol, pH 8.0 F0126 Pg (9 mg/mL), 10 mM Tris, 35 mM NaCl, 163 5 66 mM(0.5%) Glycine, 27 mM (0.5%) Mannitol, pH 8.0 F0127 Pg (9 mg/mL), 10 mMTris, 35 mM NaCl, 13 3 28.5 mM (0.5%) Arginine, 27 mM (0.5%) Mannitol,pH 8.0 F0128 Pg (9 mg/mL), 10 mM Tris, 35 mM NaCl, 119 8 26 mM (0.5%)Alanine, 27 mM (0.5%) Mannitol, pH 8.0

Example 7: Effect of Preservative on Plasminogen Aggregation

The presence of preservative, such as 0.1% phenol, has been tested inthe formulations containing 9 or 10 mg of plasminogen and 35 mM NaCl,with either 10 mM Sodium Citrate (pH 6.5), 10 mM Sodium Phosphate (pH7.2) or 10 mM Tris HCL (pH 8.0), with 0.5% arginine hydrochloride or thecombination of 0.5% arginine hydrochloride and 0.5% mannitol. Freshformulations (before lyo) were tested in this study. The particle mattercounts (PMC) have been reported in Table 8. Advantageously, the presenceof phenol has not significantly affected the particle counts.

TABLE 8 Pre-Lyo Assigned PMC_Count/mL Sample ID Sample Description 10 μm25 μm F0114 Pg (9 mg/mL), 10 mM Sodium Citrate, 35 mM NaCl, pH 6.5 3,61061 F0143 Pg (10 mg/mL), 10 mM Sodium Citrate, 35 mM NaCl, 609 8 28.5 mM(0.5%) Arginine, pH 6.5 F0144 Pg (10 mg/mL), 10 mM Sodium Citrate, 35 mMNaCl, 3,191 27 28.5 mM (0.5%) Arginine, 0.1% Phenol, pH 6.5 F0145 Pg (10mg/mL), 10 mM Sodium Citrate, 35 mM NaCl, 28.5 mM 1,538 9 (0.5%)Arginine, 27 mM (0.5%) Mannitol, 0.1% Phenol, pH 6.5 F0155 Pg (10mg/mL), 10 mM Sodium Phosphate, 35 mM NaCl, 1,097 7 28.5 mM (0.5%)Arginine, pH 7.2 F0156 Pg (10 mg/mL), 10 mM Sodium Phosphate, 35 mMNaCl, 4,370 6 28.5 mM (0.5%) Arginine, 0.1% Phenol, pH 7.2 F0157 Pg (10mg/mL), 10 mM Sodium Phosphate, 35 mM NaCl, 747 9 28.5 mM (0.5%)Arginine, 27 mM (0.5%) Mannitol, 0.1% Phenol, pH 7.2 F0158 Pg (10mg/mL), 10 mM Tris, 35 mM NaCl, 169 4 28.5 mM (0.5%) Arginine, pH 8.0F0159 Pg (10 mg/mL), 10 mM Tris, 35 mM NaCl, 168 3 28.5 mM (0.5%)Arginine, 0.1% Phenol, pH 8.0 F0160 Pg (10 mg/mL), 10 mM Tris, 35 mMNaCl, 28.5 mM 175 1 (0.5%) Arginine, 27 mM (0.5%) Mannitol, 0.1% Phenol,pH 8.0

Several preservatives were compared. Table 9 reports comparison of 0.5%CMC, 0.1% phenol and 0.5% dextran within the formations formulationscontaining 10 mg of plasminogen and 35 mM NaCl, with either 10 mM SodiumCitrate (pH 6.5), 10 mM Sodium Phosphate (pH 7.2) or 10 mM Tris HCL (pH8.0), and 0.5% arginine hydrochloride. Fresh formulations (before lyo)were tested in this study. The presence of dextran or phenol has notsignificantly affected the aggregation. However, CMC has significantlyincreased the aggregation. This is not surprising since CMC, is known toincrease the viscosity of a solution and is frequently used in thepreparation of eye drop formulation.

TABLE 9 Pre-Lyo Sample PMC Count/mL ID Sample Description 10 μm 25 μmF0119 Pg (9 mg/mL), 10 mM Sodium Phosphate, 35 mM NaCl, pH 7.2 133 6F0569 Pg (10 mg/mL) Bulk in 10 mM Na Phosphate, 35 mM NaCl, 19 1 28.5 mM(0.5%) Arginine, pH 7.2, Filtered (F0199), F/T = 1x F0570 Pg (10 mg/mL),10 mM Sodium Phosphate, 35 mM NaCl, 7,783 1 28.5 mM (0.5%) Arginine,0.5% CMC, pH 7.2 F0571 Pg (10 mg/mL), 10 mM Sodium Phosphate, 35 mMNaCl, 25 1 28.5 mM (0.5%) Arginine, 0.1% Phenol, pH 7.2 F0572 Pg (10mg/mL), 10 mM Sodium Phosphate, 35 mM NaCl, 37 1 28.5 mM (0.5%)Arginine, 0.5% Dextran, pH 7.2

Example 8: Effect of Amino Acid and its Concentration on PlasminogenAggregation

Arginine at 0.5% and 1% were tested in formulations of Plasminogen 10mg/mL, 35 mM NaCl, 10 mM Sodium Phosphate (NaPi), pH 7.2, afterlyophilisation and reconstitution. The particle counts (PMC) arereported in Table 10, which shows that both concentration of arginineare very good to maintain a low PMC value.

TABLE 10 Assign Pre-Lyo Sample Reconstitution Volume PMC Count/mL IDSample Description in dH2O (ml) (ml) 10 μm 25 μm F0211 Pg 10 mg/mL, 28mM (0.5%) Arg, 12.5 4 87 2 35 mM NaCl, 10 mM NaPi, pH 7.2 F0213 Pg 10mg/mL, 57 mM (1%) Arg, 12.5 4 124 11 35 mM NaCl, 10 mM NaPi, pH 7.2

Arginine, glycine and alanine at 1% were compared in formulations ofPlasminogen 10 mg/mL, 35 mM NaCl, 10 mM Sodium Phosphate (NaPi), pH 7.2,after lyophilisation and reconstitution. The particle counts (PMC) arereported in Table 11, which shows that arginine is better than alanineand glycine to maintain a low PMC value in the tested conditions,although all these three amino acids provide acceptable PMC values i.e.below 6000 particles equal to or greater than 10 μm per 100 mL.

TABLE 11 Assign Pre-Lyo Sample Reconstitution Volume PMC_Count/mL IDSample Description In dH2O (ml) (ml) 10 μm 25 μm F0215 Pg 10 mg/mL, 57mM (1%) Arg, 12.5 4 178 6 35 mM NaCl, 10 mM NaPi, pH 7.2 F0217 Pg 10mg/mL, 57 mM (1%) Gly, 12.5 4 881 5 35 mM NaCl, 10 mM NaPi, pH 7.2 F0219Pg 10 mg/mL, 57 mM (1%) Ala, 12.5 4 5,713 9 35 mM NaCl, 10 mM NaPi, pH7.2

Example 9: Total Percentage of Solid and Osmolality

The osmolality of several formulations of 10 mgl/mL of plasminogen andthe percentage of solid matters used to prepare said formulations arereported in Table 12. The osmolality of several formulations of 20mgl/mL of plasminogen and the percentage of solid matters used toprepare said formulations are reported in Table 13. The resultingosmolality of the tested formulations are advantageously falling withinor very close to the physiological range of osmolality. The totalpercentage of solid matters is calculated from the content ofplasminogen, tonicity modifier and bulking agent therein and is a goodindicator of the size of the resulting cake after lyophilisation.

TABLE 12 Assign Sample Total % mOsm/kg ID Sample Description Solid H₂OControl Pg Bulk (10 mg/ml) in 10 mM Na Citrate, 35 mM NaCl, pH 6.5 1.0%104 F0468 Pg (10 mg/ml) Bulk in 10 mM Na Citrate, 35 mM NaCl, pH 6.5,1.5 157 28.5 mM (0.5%) Arginine F0469 Pg (10 mg/ml) Bulk in 10 mM NaCitrate, 35 mM NaCl, pH 6.5, 2.5 209 28.5 mM (0.5%) Arginine, 54 mM (1%)Mannitol F0470 Pg (10 mg/ml) Bulk in 10 mM Na Citrate, 35 mM NaCl, pH6.5, 3.5 377 28.5 mM (0.5%) Arginine, 108 mM (2%) Mannitol F0471 Pg (10mg/ml) Bulk in 10 mM Na Citrate, 35 mM NaCl, pH 6.5, 2.5 205 28.5 mM(0.5%) Arginine, 54 mM (1%) Sorbitol F0472 Pg (10 mg/ml) Bulk in 10 mMNa Citrate, 35 mM NaCl, pH 6.5, 3.5 298 28.5 mM (0.5%) Arginine, 108 mM(2%) Sorbitol F0473 Pg (10 mg/ml) Bulk in 10 mM Na Citrate, 35 mM NaCl,pH 6.5, 57 mM 2.0 204 (1%) Arginine F0474 Pg (10 mg/ml) Bulk in 10 mM NaCitrate, 35 mM NaCl, pH 6.5, 57 mM 3.0 263 (1%) Arginine, 54 mM (1%)Mannitol F0475 Pg (10 mg/ml) Bulk in 10 mM Na Citrate, 35 mM NaCl, pH6.5, 57 mM 4.0 325 (1%) Arginine, 108 mM (2%) Mannitol F0476 Pg (10mg/ml) Bulk in 10 mM Na Citrate, 35 mM NaCl, pH 6.5, 57 mM 3.0 252 (1%)Arginine, 54 mM (1%) Sorbitol F0477 Pg (10 mg/ml) Bulk in 10 mM NaCitrate, 35 mM NaCl, pH 6.5, 57 mM 4.0 294 (1%) Arginine, 108 mM (2%)Sorbitol Control Pg Bulk (10 mg/ml) in 10 mM Na Phosphate, 35 mM NaCl,pH 7.2 1.0% 106 F0448 Pg (10 mg/ml) Bulk in 10 mM Na Phosphate, 35 mMNaCl, pH 7.2, 1.5 155 28.5 mM (0.5%) Arginine F0449 Pg (10 mg/ml) Bulkin 10 mM Na Phosphate, 35 mM NaCl, pH 7.2, 2.5 248 28.5 mM (0.5%)Arginine, 54 mM (1%) Mannitol F0450 Pg (10 mg/ml) Bulk in 10 mM NaPhosphate, 35 mM NaCl, pH 7.2, 3.5 345 28.5 mM (0.5%) Arginine, 108 mM(2%) Mannitol F0451 Pg (10 mg/ml) Bulk in 10 mM Na Phosphate, 35 mMNaCl, pH 7.2, 2.5 235 28.5 mM (0.5%) Arginine, 54 mM (1%) Sorbitol F0452Pg (10 mg/ml) Bulk in 10 mM Na Phosphate, 35 mM NaCl,pH 7.2, 3.5 37428.5 mM (0.5%) Arginine, 108 mM (2%) Sorbitol F0453 Pg (10 mg/ml) Bulkin 10 mM Na Phosphate, 35 mM NaCl, pH 7.2, 2.0 283 57 mM (1%) ArginineF0454 Pg (10 mg/ml) Bulk in 10 mM Na Phosphate, 35 mM NaCl, pH 7.2, 3.0304 57 mM (1%) Arginine, 54 mM (1%) Mannitol F0455 Pg (10 mg/ml) Bulk in10 mM Na Phosphate, 35 mM NaCl, pH 7.2, 4.0 255 57 mM (1%) Arginine, 108mM (2%) Mannitol F0456 Pg (10 mg/ml) Bulk in 10 mM Na Phosphate, 35 mMNaCl, pH 7.2, 3.0 277 57 mM (1%) Arginine, 54 mM (1%) Sorbitol F0457 Pg(10 mg/ml) Bulk in 10 mM Na Phosphate, 35 mM NaCl, pH 7.2, 4.0 284 57 mM(1%) Arginine, 108 mM (2%) Sorbitol Control Pg Bulk (10 mg/ml) in 10 mMTris-HCl, 35 mM NaCl, pH 8.0 1.00% 86 F0488 Pg (10 mg/ml) Bulk in 10 mMTris-HCl, 35 mM NaCl, pH 8.0, 28.5 mM 1.5 144 (0.5%) Arginine F0489 Pg(10 mg/ml) Bulk in 10 mM Tris-HCl, 35 mM NaCl, pH 8.0, 28.5 mM 2.5 176(0.5%) Arginine, 54 mM (1%) Mannitol F0490 Pg (10 mg/ml) Bulk in 10 mMTris-HCl, 35 mM NaCl, pH 8.0, 28.5 mM 3.5 324 (0.5%) Arginine, 108 mM(2%) Mannitol F0491 Pg (10 mg/ml) Bulk in 10 mM Tris-HCl, 35 mM NaCl, pH8.0, 28.5 mM 2.5 200 (0.5%) Arginine, 54 mM (1%) Sorbitol F0492 Pg (10mg/ml) Bulk in 10 mM Tris-HCl, 35 mM NaCl, pH 8.0, 28.5 mM 3.5 199(0.5%) Arginine, 108 mM (2%) Sorbitol F0493 Pg (10 mg/ml) Bulk in 10 mMTris-HCl, 35 mM NaCl, pH 8.0, 57 mM 2.0 186 (1%) Arginine F0494 Pg (10mg/ml) Bulk in 10 mM Tris-HCl, 35 mM NaCl, pH 8.0, 57 mM 3.0 248 (1%)Arginine, 54 mM (1%) Mannitol F0495 Pg (10 mg/ml) Bulk in 10 mMTris-HCl, 35 mM NaCl, pH 8.0, 57 mM 4.0 277 (1%) Arginine, 108 mM (2%)Mannitol F0496 Pg (10 mg/ml) Bulk in 10 mM Tris-HCl, 35 mM NaCl, pH 8.0,57 mM 3.0 239 (1%) Arginine, 54 mM (1%) Sorbitol F0497 Pg (10 mg/ml)Bulk P2146 in 10 mM Tris-HCl, 35 mM NaCl, pH 4.0 290 8.0, 57 mM (1%)Arginine, 108 mM (2%) Sorbitol

TABLE 13 Assign Sample Total % mOsm/ ID Sample Description Solid kg H₂OControl Pg Bulk (20 mg/ml) in 10 mM Na Citrate, 35 mM NaCl, pH 6.5 2.0%112 F0478 Pg (20 mg/ml) Bulk in 10 mM Na Citrate, 35 mM NaCl, pH 6.5,28.5 mM 2.5 170 (0.5%) Arginine F0479 Pg (20 mg/ml) Bulk in 10 mM NaCitrate, 35 mM NaCl, pH 6.5, 28.5 mM 3.5 245 (0.5%) Arginine, 54 mM (1%)Mannitol F0480 Pg (20 mg/ml) Bulk in 10 mM Na Citrate, 35 mM NaCl, pH6.5, 28.5 mM 4.5 307 (0.5%) Arginine, 108 mM (2%) Mannitol F0481 Pg (20mg/ml) Bulk in 10 mM Na Citrate, 35 mM NaCl, pH 6.5, 28.5 mM 3.5 255(0.5%) Arginine, 54 mM (1%) Sorbitol F0482 Pg (20 mg/ml) Bulk in 10 mMNa Citrate, 35 mM NaCl, pH 6.5, 28.5 mM 4.5 306 (0.5%) Arginine, 108 mM(2%) Sorbitol F0483 Pg (20 mg/ml) Bulk in 10 mM Na Citrate, 35 mM NaCl,pH 6.5, 57 mM 3.0 243 (1%) Arginine F0484 Pg (20 mg/ml) Bulk in 10 mM NaCitrate, 35 mM NaCl, pH 6.5, 57 mM 4.0 264 (1%) Arginine, 54 mM (1%)Mannitol F0485 Pg (20 mg/ml) Bulk in 10 mM Na Citrate, 35 mM NaCl, pH6.5, 57 mM 5.0 341 (1%) Arginine, 108 mM (2%) Mannitol F0486 Pg (20mg/ml) Bulk in 10 mM Na Citrate, 35 mM NaCl, pH 6.5, 57 mM 4.0 337 (1%)Arginine, 54 mM (1%) Sorbitol F0487 Pg (20 mg/ml) Bulk in 10 mM NaCitrate, 35 mM NaCl, pH 6.5, 57 mM 5.0 370 (1%) Arginine, 108 mM (2%)Sorbitol Control Pg Bulk P24129 (20 mg/ml) in 10 mM Na Phosphate, 35 mMNaCl, 2.0% 113 pH 7.2 F0458 Pg (20 mg/ml) Bulk in 10 mM Na Phosphate, 35mM NaCl, pH 7.2, 2.5 200 28.5 mM (0.5%) Arginine F0459 Pg (20 mg/ml)Bulk in 10 mM Na Phosphate, 35 mM NaCl, pH 7.2, 3.5 265 28.5 mM (0.5%)Arginine, 54 mM (1%) Mannitol F0460 Pg (20 mg/ml) Bulk in 10 mM NaPhosphate, 35 mM NaCl, pH 7.2, 4.5 293 28.5 mM (0.5%) Arginine, 108 mM(2%) Mannitol F0461 Pg (20 mg/ml) Bulk in 10 mM Na Phosphate, 35 mMNaCl, pH 7.2, 3.5 279 28.5 mM (0.5%) Arginine, 54 mM (1%) Sorbitol F0462Pg (20 mg/ml) Bulk in 10 mM Na Phosphate, 35 mM NaCl, pH 7.2, 4.5 31028.5 mM (0.5%) Arginine, 108 mM (2%) Sorbitol F0463 Pg (20 mg/ml) Bulkin 10 mM Na Phosphate, 35 mM NaCl, pH 7.2, 57 mM 3.0 259 (1%) ArginineF0464 Pg (20 mg/ml) Bulk in 10 mM Na Phosphate, 35 mM NaCl, pH 7.2, 57mM 4.0 350 (1%) Arginine, 54 mM (1%) Mannitol F0465 Pg (20 mg/ml) Bulkin 10 mM Na Phosphate, 35 mM NaCl, pH 7.2, 57 mM 5.0 412 (1%) Arginine,108 mM (2%) Mannitol F0466 Pg (20 mg/ml) Bulk in 10 mM Na Phosphate, 35mM NaCl, pH 7.2, 57 mM 4.0 366 (1%) Arginine, 54 mM (1%) Sorbitol F0467Pg (20 mg/ml) Bulk in 10 mM Na Phosphate, 35 mM NaCl, pH 7.2, 57 mM 5.0419 (1%) Arginine, 108 mM (2%) Sorbitol Control Pg Bulk (20 mg/ml) in 10mM Tris-HCl, 35 mM NaCl, pH 8.0 2.0% 89 F0498 Pg (20 mg/ml) Bulk in 10mM Tris-HCl, 35 mM NaCl, pH 8.0, 2.5 201 28.5 mM (0.5%) Arginine F0499Pg (20 mg/ml) Bulk in 10 mM Tris-HCl, 35 mM NaCl, pH 8.0, 3.5 260 28.5mM (0.5%) Arginine, 54 mM (1%) Mannitol F0500 Pg (20 mg/ml) Bulk in 10mM Tris-HCl, 35 mM NaCl, pH 8.0, 4.5 313 28.5 mM (0.5%) Arginine, 108 mM(2%) Mannitol F0501 Pg (20 mg/ml) Bulk in 10 mM Tris-HCl, 35 mM NaCl, pH8.0, 3.5 200 28.5 mM (0.5%) Arginine, 54 mM (1%) Sorbitol F0502 Pg (20mg/ml) Bulk in 10 mM Tris-HCl, 35 mM NaCl, pH 8.0, 4.5 319 28.5 mM(0.5%) Arginine, 108 mM (2%) Sorbitol F0503 Pg (20 mg/ml) Bulk in 10 mMTris-HCl, 35 mM NaCl, pH 8.0, 3.0 246 57 mM (1%) Arginine F0504 Pg (20mg/ml) Bulk in 10 mM Tris-HCl, 35 mM NaCl, pH 8.0, 4.0 314 57 mM (1%)Arginine, 54 mM (1%) Mannitol F0505 Pg (20 mg/ml) Bulk in 10 mMTris-HCl, 35 mM NaCl, pH 8.0, 57 mM 5.0 310 (1%) Arginine, 108 mM (2%)Mannitol F0506 Pg (20 mg/ml) Bulk in 10 mM Tris-HCl, 35 mM NaCl, pH 8.0,57 mM 4.0 282 (1%) Arginine, 54 mM (1%) Sorbitol F0507 Pg (20 mg/ml)Bulk in 10 mM Tris-HCl, 35 mM NaCl, pH 8.0, 57 mM 5.0 279 (1%) Arginine,108 mM (2%) Sorbitol

Example 10: Particle Counts (PMC)

The particle counts (PMC) for particles equal or greater than 10 μm ofthe formulations described in Table 12 are reported in Table 14; and theparticle counts (PMC) for particles equal or greater than 10 μm of theformulations described in Table 13 are reported in Table 15.

TABLE 14 Pre-Lyo Lyo-Recon Assign PMC PMC Sample Count/mL Count/mL IDSample Description 10 μm 10 μm Control Pg Bulk (10 mg/ml) in 10 mM NaCitrate, 35 mM NaCl, 379 389 pH 6.5 F0468 Pg (10 mg/ml) Bulk in 10 mM NaCitrate, 35 mM NaCl, 35 224 pH 6.5, 28.5 mM (0.5%) Arginine F0469 Pg (10mg/ml) Bulk in 10 mM Na Citrate, 35 mM NaCl, 37 136 pH 6.5, 28.5 mM(0.5%) Arginine, 54 mM (1%) Mannitol F0470 Pg (10 mg/ml) Bulk in 10 mMNa Citrate, 35 mM NaCl, 33 152 pH 6.5, 28.5 mM (0.5%) Arginine, 108 mM(2%) Mannitol F0471 Pg (10 mg/ml) Bulk in 10 mM Na Citrate, 35 mM NaCl,21 159 pH 6.5, 28.5 mM (0.5%) Arginine, 54 mM (1%) Sorbitol F0472 Pg (10mg/ml) Bulk in 10 mM Na Citrate, 35 mM NaCl, 94 258 pH 6.5, 28.5 mM(0.5%) Arginine, 108 mM (2%) Sorbitol F0473 Pg (10 mg/ml) Bulk in 10 mMNa Citrate, 35 mM NaCl, 45 342 pH 6.5, 57 mM (1%) Arginine F0474 Pg (10mg/ml) Bulk in 10 mM Na Citrate, 35 mM NaCl, 61 218 pH 6.5, 57 mM (1%)Arginine, 54 mM (1%) Mannitol F0475 Pg (10 mg/ml) Bulk in 10 mM NaCitrate, 35 mM NaCl, 129 138 pH 6.5, 57 mM (1%) Arginine, 108 mM (2%)Mannitol F0476 Pg (10 mg/ml) Bulk in 10 mM Na Citrate, 35 mM NaCl, 112401 pH 6.5, 57 mM (1%) Arginine, 54 mM (1%) Sorbitol F0477 Pg (10 mg/ml)Bulk in 10 mM Na Citrate, 35 mM NaCl, 155 159 pH 6.5, 57 mM (1%)Arginine, 108 mM (2%) Sorbitol Control Pg Bulk (10 mg/ml) in 10 mM NaPhosphate, 35 mM NaCl, 2,400 3,819 pH 7.2 F0448 Pg (10 mg/ml) Bulk in 10mM Na Phosphate, 35 mM NaCl, 135 198 pH 7.2, 28.5 mM (0.5%) ArginineF0449 Pg (10 mg/ml) Bulk in 10 mM Na Phosphate, 35 mM NaCl, 171 104 pH7.2, 28.5 mM (0.5%) Arginine, 54 mM (1%) Mannitol F0450 Pg (10 mg/ml)Bulk in 10 mM Na Phosphate, 35 mM NaCl, 267 254 pH 7.2, 28.5 mM (0.5%)Arginine, 108 mM (2%) Mannitol F0451 Pg (10 mg/ml) Bulk in 10 mM NaPhosphate, 35 mM NaCl, 122 133 pH 7.2, 28.5 mM (0.5%) Arginine, 54 mM(1%) Sorbitol F0452 Pg (10 mg/ml) Bulk in 10 mM Na Phosphate, 35 mMNaCl, 214 176 pH 7.2, 28.5 mM (0.5%) Arginine, 108 mM (2%) SorbitolF0453 Pg (10 mg/ml) Bulk in 10 mM Na Phosphate, 35 mM NaCl, 37 262 pH7.2, 57 mM (1%) Arginine F0454 Pg (10 mg/ml) Bulk in 10 mM Na Phosphate,35 mM NaCl, 63 84 pH 7.2, 57 mM (1%) Arginine, 54 mM (1%) Mannitol F0455Pg (10 mg/ml) Bulk in 10 mM Na Phosphate, 35 mM NaCl, 109 150 pH 7.2, 57mM (1%) Arginine, 108 mM (2%) Mannitol F0456 Pg (10 mg/ml) Bulk in 10 mMNa Phosphate, 35 mM NaCl, 86 169 pH 7.2, 57 mM (1%) Arginine, 54 mM (1%)Sorbitol F0457 Pg (10 mg/ml) Bulk in 10 mM Na Phosphate, 35 mM NaCl, 85229 pH 7.2, 57 mM (1%) Arginine, 108 mM (2%) Sorbitol Control Pg Bulk(10 mg/ml) in 10 mM Tris-HCl, 35 mM NaCl, pH 8.0 3,341 4,007 F0488 Pg(10 mg/ml) Bulk in 10 mM Tris-HCl, 35 mM NaCl, pH 8.0, 150 414 28.5 mM(0.5%) Arginine F0489 Pg (10 mg/ml) Bulk in 10 mM Tris-HCl, 35 mM NaCl,pH 8.0, 151 387 28.5 mM (0.5%) Arginine, 54 mM (1%) Mannitol F0490 Pg(10 mg/ml) Bulk in 10 mM Tris-HCl, 35 mM NaCl, pH 8.0, 91 528 28.5 mM(0.5%) Arginine, 108 mM (2%) Mannitol F0491 Pg (10 mg/ml) Bulk in 10 mMTris-HCl, 35 mM NaCl, pH 8.0, 240 589 28.5 mM (0.5%) Arginine, 54 mM(1%) Sorbitol F0492 Pg (10 mg/ml) Bulk in 10 mM Tris-HCl, 35 mM NaCl, pH8.0, 120 507 28.5 mM (0.5%) Arginine, 108 mM (2%) Sorbitol F0493 Pg (10mg/ml) Bulk in 10 mM Tris-HCl, 35 mM NaCl, pH 8.0, 103 229 57 mM (1%)Arginine F0494 Pg (10 mg/ml) Bulk in 10 mM Tris-HCl, 35 mM NaCl, pH 8.0,90 507 57 mM (1%) Arginine, 54 mM (1%) Mannitol F0495 Pg (10 mg/ml) Bulkin 10 mM Tris-HCl, 35 mM NaCl, pH 8.0, 67 256 57 mM (1%) Arginine, 108mM (2%) Mannitol F0496 Pg (10 mg/ml) Bulk in 10 mM Tris-HCl, 35 mM NaCl,pH 8.0, 131 375 57 mM (1%) Arginine, 54 mM (1%) Sorbitol F0497 Pg (10mg/ml) Bulk in 10 mM Tris-HCl, 35 mM NaCl, pH 8.0, 62 456 57 mM (1%)Arginine, 108 mM (2%) Sorbitol

TABLE 15 Lyo- Pre-Lyo Recon Assign PMC PMC Sample Count/mL Count/mL IDSample Description 10 μm 10 μm Control Pg Bulk (20 mg/ml) in 10 mM NaCitrate, 35 mM NaCl, pH 6.5 1100 793 F0478 Pg (20 mg/ml) Bulk in 10 mMNa Citrate, 35 mM NaCl, pH 6.5, 171 358 28.5 mM (0.5%) Arginine F0479 Pg(20 mg/ml) Bulk in 10 mM Na Citrate, 35 mM NaCl, pH 6.5, 118 572 28.5 mM(0.5%) Arginine, 54 mM (1%) Mannitol F0480 Pg (20 mg/ml) Bulk in 10 mMNa Citrate, 35 mM NaCl, pH 6.5, 132 515 28.5 mM (0.5%) Arginine, 108 mM(2%) Mannitol F0481 Pg (20 mg/ml) Bulk in 10 mM Na Citrate, 35 mM NaCl,pH 6.5, 96 427 28.5 mM (0.5%) Arginine, 54 mM (1%) Sorbitol F0482 Pg (20mg/ml) Bulk in 10 mM Na Citrate, 35 mM NaCl, pH 6.5, 101 585 28.5 mM(0.5%) Arginine, 108 mM (2%) Sorbitol F0483 Pg (20 mg/ml) Bulk in 10 mMNa Citrate, 35 mM NaCl, pH 6.5, 132 286 57 mM (1%) Arginine F0484 Pg (20mg/ml) Bulk in 10 mM Na Citrate, 35 mM NaCl, pH 6.5, 115 537 57 mM (1%)Arginine, 54 mM (1%) Mannitol F0485 Pg (20 mg/ml) Bulk in 10 mM NaCitrate, 35 mM NaCl, pH 6.5, 154 362 57 mM (1%) Arginine, 108 mM (2%)Mannitol F0486 Pg (20 mg/ml) Bulk in 10 mM Na Citrate, 35 mM NaCl, pH6.5, 114 535 57 mM (1%) Arginine, 54 mM (1%) Sorbitol F0487 Pg (20mg/ml) Bulk in 10 mM Na Citrate, 35 mM NaCl, pH 6.5, 194 384 57 mM (1%)Arginine, 108 mM (2%) Sorbitol Control Pg Bulk (20 mg/ml) in 10 mM NaPhosphate, 35 mM NaCl, 3,784 2,619 pH 7.2 F0458 Pg (20 mg/ml) Bulk in 10mM Na Phosphate, 35 mM NaCl, 75 398 pH 7.2, 28.5 mM (0.5%) ArginineF0459 Pg (20 mg/ml) Bulk in 10 mM Na Phosphate, 35 mM NaCl, 96 193 pH7.2, 28.5 mM (0.5%) Arginine, 54 mM (1%) Mannitol F0460 Pg (20 mg/ml)Bulk in 10 mM Na Phosphate, 35 mM NaCl, 128 198 pH 7.2, 28.5 mM (0.5%)Arginine, 108 mM (2%) Mannitol F0461 Pg (20 mg/ml) Bulk in 10 mM NaPhosphate, 35 mM NaCl, 123 659 pH 7.2, 28.5 mM (0.5%) Arginine, 54 mM(1%) Sorbitol F0462 Pg (20 mg/ml) Bulk in 10 mM Na Phosphate, 35 mMNaCl, 103 317 pH 7.2, 28.5 mM (0.5%) Arginine, 108 mM (2%) SorbitolF0463 Pg (20 mg/ml) Bulk in 10 mM Na Phosphate, 35 mM NaCl, 93 270 pH7.2, 57 mM (1%) Arginine F0464 Pg (20 mg/ml) Bulk in 10 mM Na Phosphate,35 mM NaCl, 99 386 pH 7.2, 57 mM (1%) Arginine, 54 mM (1%) MannitolF0465 Pg (20 mg/ml) Bulk in 10 mM Na Phosphate, 35 mM NaCl, 139 172 pH7.2, 57 mM (1%) Arginine, 108 mM (2%) Mannitol F0466 Pg (20 mg/ml) Bulkin 10 mM Na Phosphate, 35 mM NaCl, 67 681 pH 7.2, 57 mM (1%) Arginine,54 mM (1%) Sorbitol F0467 Pg (20 mg/ml) Bulk in 10 mM Na Phosphate, 35mM NaCl, 93 261 pH 7.2, 57 mM (1%) Arginine, 108 mM (2%) SorbitolControl Pg Bulk (20 mg/ml) in 10 mM Tris-HCl, 35 mM NaCl, pH 8.0 6,3157,123 F0498 Pg (20 mg/ml) Bulk in 10 mM Tris-HCl, 35 mM NaCl, pH 8.0,629 3,382 28.5 mM (0.5%) Arginine F0499 Pg (20 mg/ml) Bulk in 10 mMTris-HCl, 35 mM NaCl, pH 8.0, 1329 1,596 28.5 mM (0.5%) Arginine, 54 mM(1%) Mannitol F0500 Pg (20 mg/ml) Bulk in 10 mM Tris-HCl, 35 mM NaCl, pH8.0, 1007 896 28.5 mM (0.5%) Arginine, 108 mM (2%) Mannitol F0501 Pg (20mg/ml) Bulk in 10 mM Tris-HCl, 35 mM NaCl, pH 8.0, 649 1,401 28.5 mM(0.5%) Arginine, 54 mM (1%) Sorbitol F0502 Pg (20 mg/ml) Bulk in 10 mMTris-HCl, 35 mM NaCl, pH 8.0, 793 684 28.5 mM (0.5%) Arginine, 108 mM(2%) Sorbitol F0503 Pg (20 mg/ml) Bulk in 10 mM Tris-HCl, 35 mM NaCl, pH8.0, 459 703 57 mM (1%) Arginine F0504 Pg (20 mg/ml) Bulk in 10 mMTris-HCl, 35 mM NaCl, pH 8.0, 57 mM 505 608 (1%) Arginine, 54 mM (1%)Mannitol F0505 Pg (20 mg/ml) Bulk P2147 in 10 mM Tris-HCl, 35 mM NaCl,155 382 pH 8.0, 57 mM (1%) Arginine, 108 mM (2%) Mannitol F0506 Pg (20mg/ml) Bulk in 10 mM Tris-HCl, 35 mM NaCl, pH 8.0, 57 mM 213 898 (1%)Arginine, 54 mM (1%) Sorbitol F0507 Pg (20 mg/ml) Bulk in 10 mMTris-HCl, 35 mM NaCl, pH 8.0, 57 mM 101 431 (1%) Arginine, 108 mM (2%)Sorbitol

Pictures of vials are shown in FIGS. 12, 13 and 14, which containsformulations F0498, F0449 and F0459, respectively, as representativeexamples of the appearance of the lyophilized formulations reported inTables 12-1′5.

Example 10: Reversible Plasminogen Aggregation

Plasminogen aggregation is reversible as demonstrated by this study.Frozen Plasminogen samples (Pg Bulk) 5 mg/ml, dialyzed in dH2O andheavily precipitated, were thawed, filtered through 1.2 μm syringefilter, and used as starting material for control (Pg control). Aliquotsof 2 ml Pg control were added to glass vials, pre-spiked with bufferstocks of 1 M Na Citrate pH 6.5 (Sample 1), 0.5 M Na Phosphate pH 7.2(Sample 2) or 1 M Tris-HCl pH 8.0 (Sample 3) to obtain final 10 mMbuffer concentration at each pH, respectively. Samples 1, 2 and 3 areshown in FIG. 15, where the non-spiked Pg control is Sample 0. Samples1, 2 and 3 were further spiked with stock solutions of 5 M NaCl and 0.8M Arginine to obtain a final concentration of 35 mM NaCl and 28.5 mM(0.5%) Arg in each sample and are shown in FIG. 16. Turbidity standardsof 0.02, 20, 100, 800 NTU respectively, were used in FIGS. 15 and 16, asreference. It can be noted from FIG. 15 that pH adjustment has dissolveda considerable amount of aggregates. In FIG. 16, the Samples 1, 2 and 3are rendered transparent by the addition of Arginine and NaCl and noaggregate can be visually detected.

The turbidity of Samples 0, 1, 2 and 3 of FIG. 16 were measured byreading the Optical Density (OD) at 550 nm using standard calibrationwith turbidity reference standards shown in FIGS. 15 and 16. Theturbidity is reported in Nephelometric Turbidity Units (NTU) in Table16. Aliquots of 1 ml of each sample was measured before (T=0) and afterspiking the samples 1, 2 and 3 with buffer, NaCl and Arginine at 0.1hour, 17 hours, 21 hours and 24 hours. Disaggregation is rapid as it canbe noted at only 0.1 hour after spiking, and it is maintained over 24hours.

TABLE 16 Assigned Sample Turbidity (NTU by OD 550) ID Sample DescriptionT = 0 T = 0.1 h T = 17 h T = 21 h T = 24 h F0138 Pg Bulk (5 mg/ml),Conc. 2 × dH2O exchanged 1008 1008 1209 858 372 F0139 Pg Bulk (5 mg/ml)in 10 mM Sodium Citrate, 1008 30 37 39 60 35 mM NaCl, 28.5 mM (0.5%)Arginine, pH 6.5 F0140 Pg Bulk (5 mg/ml) in 10 mM Sodium 1008 49 60 68100 Phosphate, 35 mM NaCl, 28.5 mM (0.5%) Arginine, pH 7.2 F0141 Pg Bulk(5 mg/ml) in 10 mM Tris, 1008 49 56 66 92 35 mM NaCl, 28.5 mM (0.5%)Arginine, pH 8.0

Example 11: Effect of Amino Acid Over Time on Composition Turbidity

Turbidity has been measured in compositions containing 10 mg plasminogenand 35 mM NaCl with 0.5% glycine, arginine, alanine and without aminoacid, at pH 6.5 in sodium citrate buffer (FIG. 17), pH 7.2 in sodiumphosphate buffer (FIG. 18), and at pH 8.0 in Tris buffer (FIG. 19).

The compositions tested in FIGS. 17, 18 and 19 were reproduced inpresence of 0.5% mannitol as a bulking agent, and reported in FIGS. 20,21 and 22, respectively.

In all cases, the presence of the amino acid provides a stabilizingeffect and maintain or lower the turbidity. Comparing the type of aminoacids, arginine shows the best stabilizing effect and lower theturbidity at all tested pH and in presence and absence of bulking agent,i.e. 0.5% mannitol.

Example 12: Composition of Elevated Concentration of Plasminogen

Compositions containing a high concentration of plasminogen have beenstudied. The PMC counts for particles of 10 μm or greater, and of 25 μmor greater have been reported in Table 17 for compositions of 20 mg/mlplasminogen in 10 mM or 100 mM sodium citrate, 35 mM NaCl, at pH6.5,with and without 1% arginine, and compositions of 37 and 56 mg/mlplasminogen in 100 mM sodium citrate, 35 mM NaCl, at pH6.5, with 1%arginine.

Table 17 shows that the formulations of the present inventionadvantageously allow the preparation of compositions comprising elevatedconcentrations of plasminogen (37 and 56 mg/ml) without causinginacceptable level of particles,

TABLE 17 Assign PMC_Count/Vial Sample ID Sample Description 10 μm 25 μmP24138 Pg (20 mg/ml) Bulk in 10 mM Na Citrate, 35 mM NaCl, pH 6.5 983 8F0483 Pg (20 mg/ml) Bulk in 10 mM Na Citrate, 35 mM NaCl, pH 6.5, 525 057 mM (1%) Arginine F0614 Pg (20 mg/ml) Bulk in 10 mM Na Citrate, 100 mMNaCl, pH 6.5, 667 33 57 mM (1%) Arginine F0615 Pg (37 mg/ml) Bulk in 10mM Na Citrate, 100 mM NaCl, pH 6.5, 325 33 57 mM (1%) Arginine F0616 Pg(56 mg/ml) Bulk in 10 mM Na Citrate, 100 mM NaCl, pH 6.5, 292 17 57 mM(1%) Arginine

Example 13: Plasminogen Activity

The plasminogen activity for each formulation exemplified herein wastested and compared to the plasminogen activity of the initialpreparation or bulk preparation (Pg bulk). It has been noted that theplasminogen activity is not affected by any of these formulations.

1. A liquid pharmaceutical composition for administration to a subject,said composition having an osmolality of 180 mOsm to 350 mOsm andcomprising: plasminogen, or a biologically active variant thereof, in aconcentration from 0.01 mg/ml to 80 mg/ml; a tonicity modifier, whichcontains sodium, at a concentration of 30 mM to 250 mM; and astabilising agent, which is arginine or glycine, in a concentration from20 mM to 200 mM; wherein the composition has a pH of 3.0 to 10.0.
 2. Thepharmaceutical composition of claim 1, wherein the stabilising agent isarginine.
 3. The pharmaceutical composition of claim 1, wherein thestabilising agent is glycine.
 4. The pharmaceutical composition of claim1, wherein the pH is from 5.0 to 8.0.
 5. The pharmaceutical compositionof claim 4, wherein the pH is from 6.0 to 8.0.
 6. The pharmaceuticalcomposition of claim 4, wherein the pH is from 6.5 to 7.5.
 7. Thepharmaceutical composition of claim 1, wherein the concentration of theplasminogen or biologically active variant thereof is from 5 mg/ml to 60mg/ml.
 8. The pharmaceutical composition of claim 7, wherein theconcentration of the plasminogen or biologically active variant thereofis 40, 30, 20, 10 or 5 mg/ml.
 9. The pharmaceutical composition of claim1, wherein the stabilising agent is in a concentration of 25 mM to 75mM.
 10. The pharmaceutical composition of claim 1, wherein the tonicitymodifier is sodium chloride.
 11. The pharmaceutical composition of claim1, wherein the tonicity modifier is present in a concentration of 30 mMto 100 mM.
 12. The pharmaceutical composition of claim 1, furthercomprising a non-reducing sugar, which is sucrose.
 13. Thepharmaceutical composition of claim 1, wherein said plasminogen or abiologically active variant thereof is Glu-plasminogen.
 14. Thepharmaceutical composition of claim 1, wherein the composition containsan amount of particles equal to or greater than 10 μm that is lower than2000 particles per 100 ml.
 15. The pharmaceutical composition of claim14, wherein the composition contains an amount of particles equal to orgreater than 10 μm that is lower than 1000 particles per 100 ml.
 16. Thepharmaceutical composition of claim 1, which is suitable forintravenous, subcutaneous, topical, intradermal, ophthalmic and/orintramuscular administration.
 17. The pharmaceutical composition ofclaim 1, which is a reconstituted composition.
 18. A method for treatinga wound, tympanic membrane perforation, a periodontal wound, aninfectious disease, posterior vitreous detachment (PVD), a diabeticulcer, plasminogen-deficiency, a thrombolysis, reperfusion injury totissue, ischemia, infarction, or brain edema, or for increasing ormaintaining oral health, improving microcirculation, or modulating thecomplement pathway, wherein said method comprises administering to asubject the pharmaceutical composition of claim
 1. 19. The method ofclaim 18, used for treating of a wound in a subject.
 20. The method ofclaim 18, used for treating a plasminogen-deficiency in a subject. 21.The method of claim 18, used for treating a diabetic ulcer in a subject.